TEMPERATURE ADAPTATION IN ENZYMES FROM POIKILOTHERMS:
ACETYLCHOLINESTERASES IN THE NERVOUS SYSTEM OF FISHES
by
JOHN BALDWIN
B.Sc.
M.Sc,
(Hons.), Monash U n i v e r s i t y , 1965
Monash U n i v e r s i t y , 196,9
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
i n the Department
of
Zoology
We a c c e p t t h i s t h e s i s as conforming to the
r e q u i r e d standard
THE UNIVERSITY OF BRITISH COLUMBIA
A p r i l 1970
In
presenting
this
an a d v a n c e d d e g r e e
the
I
Library
further
for
agree
in
at
University
the
make
tha
it
partial
freely
permission
this
representatives.
thesis
for
It
financial
for
of
gain
^-o-e»li
The U n i v e r s i t y o f B r i t i s h
V a n c o u v e r 8 , Canada
of
of
Columbia,
British
for
extensive
by
the
Columbia
shall
not
the
requirements
reference
copying of
Head o f
is understood that
written permission.
Department
fulfilment
available
s c h o l a r l y p u r p o s e s may be g r a n t e d
by h i s
of
shall
thesis
I
agree
and
this
be a l l o w e d
that
study.
thesis
my D e p a r t m e n t
copying or
for
or
publication
w i t h o u t my
i
ABSTRACT
The
e f f e c t s of temperature
(AChE) f r o m
if
the n e r v o u s
such compensatory
acclimation
displayed
At
level
trout
(Salmo
unaffected
temperature
substrate
hydrolysis
by a s s a y temperature
be
and
c o n c e n t r a t i o n s the
b y AChE f r o m
e e l remains
these animals.
a t temperatures
Plots
rainbow
relatively
U shaped
close
ranges
o f Km
versus
c u r v e s w i t h minimum
t o t h e minimum
temperature.
I t i s proposed that
reaction
rate
i s a c h i e v e d throughout the h a b i t a t
range by
temperature
directed
as
observed
o v e r the temperature
f o r t h e s e enzymes y i e l d
values occurring
could
thermal
function.
g a i r d n e r i i ) and e l e c t r i c
n o r m a l l y e x p e r i e n c e d by
Km
o f enzyme
(ACh)
determine
t h e r m a l accommodation,
system
probable p h y s i o l o g i c a l
of a c e t y l c h o l i n e
to
a d a p t a t i o n to temperature
by t h i s p h y s i o l o g i c a l
a t the
acetylcholinesterase
o f f i s h were s t u d i e d
phenomena as
and e v o l u t i o n a r y
interpreted
rate
system
upon
habitat
t h e r m a l accommodation o f
temperature
changes i n enzyme-substrate
affinity.
Thermal
speckled
trout
acclimation
(Salvelinus
namaychus) i s a c c o m p a n i e d
proportions
displaying
Since
two
of
o f two
trout,
f o n t i n a l i s ) and
by a l t e r a t i o n s
and
lake
probably i n
trout
(Salvelinus
i n the r e l a t i v e
e l e c t r o p h o r e t i c a l l y d i s t i n c t AChE
d i f f e r e n t a n d a d a p t i v e Km-temperature
the minimum Km
rainbow
i n rainbow
variants
relationships.
v a l u e s and e n e r g i e s o f a c t i v a t i o n o f
t r o u t enzymes a r e s i m i l a r , and
the
the s p e c i f i c a c t i v i t i e s
t h e enzymes a r e e s s e n t i a l l y i d e n t i c a l f o l l o w i n g
acclimation
of
fish
to
2°
and
AChE a c t i v i t y may
However, the
pH,
ionic
17°C,
not
i t i s suggested that
occur at
possibility
acclimation
p r o c e s s may
eel
and
the
the
evolutionary
different
Antarctic
of
reaction
Shifts
i n the
by
central
fish
o v e r the
the
electric
indicate
i n species
temperature
thermal
range n o r m a l l y
r e l a t i o n s h i p during
AChE enzymes c o u l d
be
encountered.
speciation
n e r v o u s s y s t e m were c o n s i d e r e d and
a
into
mechanisms
the
trout
hypothesis
i n v o l v i n g h y b r i d i z a t i o n between f i s h
p o p u l a t i o n s was
trout
observed that hybrids
inter-species
crosses.
between s p e c k l e d and
lake
I t was
trout contained a greater
e l e c t r o p h o r e t i c a l l y d i s t i n c t AChE v a r i a n t s
and
further,
complexes
the
the
presence of
i n rainbow,
original
incorporation
of m u l t i p l e
rainbow t r o u t
probably occurred
divergence of
these
It
substrate
directed
three
trout
number
the
with
formed
of
parent
c o n t r o l l e d AChE
indicated
that
AChE enzymes i n t o
p r i o r to
the
evolutionary
species.
i s c o n c l u d e d from t h i s
a f f i n i t y with
lake
tested
than d i d e i t h e r
s i m i l a r thermally
s p e c k l e d and
are
following
Possible
incorporated
a
accommodation
c h a n g e s i n enzyme c o n f o r m a t i o n
amino a c i d s u b s t i t u t i o n s .
that
inhabiting
i s b a s e d upon s e l e c t i o n f o r
allow
as
rates.
Trematomus b o r c h g r e v i n k i
Km-temperature
i n terms o f
w h i c h two
reaction
a d a p t a t i o n o f AChE f u n c t i o n
accumulation of
i n such f a c t o r s
AChE enzymes f r o m r a i n b o w t r o u t ,
relationship that w i l l
rate
interpreted
the
to s t a b i l i z e
thermal environments
Km-temperature
changes
of
temperatures.
membrane l i p i d s w h i c h accompany
act
Comparisons o f
compensation
different acclimation
remains t h a t
e n v i r o n m e n t and
rate
study that
temperature,
and
p r o d u c t i o n o f enzyme v a r i a n t s
the
c h a n g e s i n enzymetemperature
displaying
adaptive
Km-temperature
for controlling
functions
relationships,
are both
i m p o r t a n t mechanisms
c a t a l y t i c a c t i v i t y i n a n enzyme s y s t e m
o v e r a wide range
of
temperatures.
which
iv
TABLE OF
CONTENTS
Page
Abstract
i
List
of
Tables
v i i
List
of
Figures
viii
Acknowledgements
x
Introduction
1
1.
Statement of
2.
T h e r m a l Accommodation and
in
3.
the
the
Central
Problem
1
Thermal
N e r v o u s System o f
Role of A c e t y l c h o l i n e s t e r a s e
Acclimation
Poikilotherms
i n Nerve
Transmission
4.
Importance o f
Central
3
5
C h o l i n e r g i c Mechanisms i n
Nervous
the
System
6
Methods
12
1.
Experimental Animals
12
2.
Enzyme P r e p a r a t i o n s
13
a.
Preparation
of
Rainbow T r o u t
Brain
Acetylcholinesterase
b.
Preparation
Trout
13
of A c e t y l c h o l i n e s t e r a s e
B r a i n and
Spinal
Cord f o r
from
Electro-
phoresis
14
c.
Electric
Eel Acetylcholinesterase
d.
Preparation
of
Trematomus
14
borchgrevinki
Brain Acetylcholinesterase
Activity
15
3.
Assay of A c e t y l c h o l i n e s t e r a s e
15
4.
Gel
Electrophoresis
15
5.
Protein Determinations
16
Page
6.
Sucrose Gradient
7.
Ultraviolet
Results
1.
Centrifugation
Difference
16
Spectra
18
a.
Introduction
18
b.
Method
18
and D i s c u s s i o n
20
Partial
Purification
o f Rainbow T r o u t
Brain
Acetylcholinesterase
2.
Characterization of Acetylcholinesterase
Rainbow T r o u t
Central
a.
Introduction
b.
Multiple
in
c.
Nervous
Forms o f
Substrate
System
20
Acetylcholinesterase
Central
S p e c i f i c i t y and
o f Rainbow T r o u t
Nervous
System
20
Inhibition
Brain
Acetyl-
cholinesterase
22
d.
Effect
27
e.
Sucrose Gradient
Trout
o f pH on AChE A c t i v i t y
C e n t r i f u g a t i o n o f Rainbow
Brain Acetylcholinesterase
27
Characterization of Acetylcholinesterase
Trematomus b o r c h g r e v i n k i
4.
from the
20
t h e Rainbow T r o u t
Studies
3.
20
Effect
a.
Brain
o f A s s a y T e m p e r a t u r e upon
of A c e t y l c h o l i n e
Effect
Hydrolysis
30
the K i n e t i c s
by A c e t y l c h o l i n e s t e r a s e
Effect
30
o f T e m p e r a t u r e on t h e Maximum
V e l o c i t y of Acetylcholinesterase
b.
from
Hydrolysis
30
o f A s s a y T e m p e r a t u r e on Enzyme-
Substrate
Affinity
35
vi
Page
c.
R e l a t i o n s h i p between T h e r m a l l y Induced
Changes i n Km a n d S t r u c t u r a l C o n f o r m a t i o n
of E l e c t r i c
5.
Eel Acetylcholinesterase
T h e r m a l Accommodation,
Evolutionary
Thermal A c c l i m a t i o n
Adaptation
Acetylcholinesterase
45
and
t o Temperature f o r
from t h e Nervous
System o f
Fish.
53
a.
Thermal Accommodation
53
b.
Thermal A c c l i m a t i o n
59
(i)
Adjustment
o f the thermal
accommodation
(ii)
c.
range
59
Rate c o m p e n s a t i o n o f AChE a c t i v i t y
E v o l u t i o n a r y A d a p t a t i o n t o Temperature
(i)
68
Adjustment o f the thermal
accommodation
(ii)
60
range
68
E v o l u t i o n o f the rainbow t r o u t b r a i n
AChE complex
( i i i ) Regulation
the
69
o f the c o m p o s i t i o n o f
t r o u t b r a i n AChE complex
thermal a c c l i m a t i o n
during
73
Summary
76
Abbreviations
80
Literature
81
Cited
L I S T OF
Partial
Trout
Purification
TABLES
o f AChE
Rainbow
Brain
Summation E x p e r i m e n t s w i t h
and
from
the E f f e c t
Choline
of Inhibitors
on the
H y d r o l y s i s o f ACh b y Rainbow T r o u t
Electric
E e l AChEs
Apparent
Energies
the
at
Rainbow T r o u t
Several
of A c t i v a t i o n
and E l e c t r i c
E e l AChEs
Temperatures
o f Temperature
Trematomus
upon t h e Km a n d Rate
f o r B r a i n AChE
o f t h e Rate
Change a n d
o f ACh H y d r o l y s i s
Trematomus
1 Q
a t ConcentraKm f o r
Electric Eel
borchgrevinki
o f S a l t s o n t h e Km a n d Rate o f
Hydrolysis
o f ACh b y AChE
Acclimated
Rainbow
Specific Activities
from 2°C
Trout
o f B r a i n AChE
n
Rainbow T r o u t A c c l i m a t e d
for
Q
o f ACh A p p r o a c h i n g t h e Minimum
AChEs f r o m Rainbow T r o u t ,
Effect
from
borchgrevinki
R e l a t i o n s h i p b e t w e e n Km
and
Trout,
E e l a n d Trematomus
o f ACh H y d r o l y s i s
tions
Km
o f ACh f o r AChEs f r o m Rainbow
Electric
Effect
and
(Ea) f o r
R a t e s o f ACh H y d r o l y s i s a t Minimum
Levels
Esters
35 Days
to 2
from
o
a n d 17 C
V l l l
L I S T OF
FIGURES
Figure
1
Facing
Page
R e s o l u t i o n o f Rainbow T r o u t B r a i n AChEs
by A c r y l a m i d e
2
Substrate
Substrate
Rainbow T r o u t
24
S p e c i f i c i t y o f AChE f r o m 17°C
Acclimated
4
23
S p e c i f i c i t y o f AChE f r o m 2 ° C
Acclimated
3
Gel Disc Electrophoresis
Rainbow T r o u t
25
I n f l u e n c e o f pH on t h e A c t i v i t y
o f Rainbow
T r o u t B r a i n AChEs
5
Sucrose
Gradient
28
C e n t r i f u g a t i o n o f Rainbow
T r o u t a n d E l e c t r i c E e l AChEs
6
Arrhenius
29
P l o t s o f AChE A c t i v i t y
f o r the
Rainbow T r o u t a n d E l e c t r i c E e l Enzymes
7
Effect
33
o f A s s a y T e m p e r a t u r e o n t h e Km o f
°
AChE f o r AChEs f r o m
17
o
and 2 C A c c l i m a t e d
Rainbow T r o u t
8
Effect
for
9
38
o f A s s a y T e m p e r a t u r e o n t h e Km o f ACh
E l e c t r i c E e l AChE
Effect
39
o f A s s a y T e m p e r a t u r e o n t h e Km o f
ACh f o r Trematomus b o r c h g r e v i n k i AChE.
Lineweaver-Burk P l o t s a t 2° and 10°C.
10
Sucrose
Gradient
Electric
11
Effect
Sedimentation
Profiles
43
of
E e l AChE a t 1 5 ° , 2 5 ° a n d 3 3 ° C
48
o f T e m p e r a t u r e o n t h e Km o f ACh f o r
Electric
E e l AChE A s s a y e d i n t h e C e n t r i f u g a -
t i o n Medium. L i n e w e a v e r - B u r k
25° and 33°C.
P l o t s a t 15°,
49
E f f e c t o f Temperature upon the Sedimentation
Behaviour o f E l e c t r i c E e l AChE
Ultraviolet
D i f f e r e n c e Spectra o f E l e c t r i c
E e l AChE as a F u n c t i o n of Temperature
E f f e c t o f Assay Temperature on the Km o f ACh
f o r AChEs from Rainbow Trout, E l e c t r i c E e l and
Trematomus
ACh S a t u r a t i o n Curves o f E l e c t r i c E e l AChE
at
o
o
o
15 , 25 and 40 C
ACh S a t u r a t i o n Curves o f Trematomus
b o r c h g r e v i n k i AChE a t 2° and 10°C
ACh S a t u r a t i o n Curves o f 2°C A c c l i m a t e d
o
o
o
o
Rainbow Trout AChE a t 0 , 2 , 12 and 18 C
R e s o l u t i o n o f B r a i n AChEs from
Trout,
Speckled
Lake Trout and Splake by Acrylamide
Gel D i s c E l e c t r o p h o r e s i s
X
ACKNOWLEDGEMENTS
I am e s p e c i a l l y g r a t e f u l
George
Somero f o r a d v i c e a n d e n c o u r a g e m e n t t h r o u g h o u t t h e
course of t h i s study.
I would l i k e
f o r use o f the u l t r a c e n t r i f u g e
available
Dr.
t o D r s . P e t e r Hochachka and
t o thank
the Cary spectrophotometer.
J.
I. am? g r a t e f u l t o
F. E . J . F r y a n d T e r r y McFadden a t t h e U n i v e r s i t y
speckled,
lake and splake t r o u t
t h e P i n a s k Lake r a i n b o w
E. P h i l l i p s
critical
experiments,
trout.
and to T e r r y
Gjernes
To D r s . G. I . Drujrimond,
a n d D. J . R a n d a l l go s p e c i a l
reading of this thesis.
o f Toronto
f o r s e t t i n g up t h e
thanks
I would a l s o
my w i f e Wendy, a n d M r s . LouAnne Moon f o r t y p i n g
drafts.
Green
a n d D r . S. EL Z b a r s k y who made
Laboratory f o r Experimental Limnology
for
Dr. B e v e r l y
like
f o r their
t o thank
innumerable
1
INTRODUCTION
1.
Statement o f the
The
ability
Problem
of poikilotherms
thermal environments
out r e l a t i v e l y
and
problems
thesis
to describe
t h e i r environment.
claims
The
several
reason f o r t h i s
t h e s e c o n c e p t s may
term
of a function
a particular
Thermal
be
to
terms, b u t r a t h e r t o
are o f t e n used l o o s e l y
give
i n the
level,
so
extended to the m o l e c u l a r l e v e l .
"thermal accommodation" w i l l
temperature
refer
range,
t o the
the t h e r m a l accommodation
accommodation o c c u r s i n s t a n t a n e o u s l y and
"Thermal
ability
acclimation" describes
the a b i l i t y
intrinsic
The
physiological
of a
temperature
a p e r i o d o f exposure o f an i n d i v i d u a l
thermal regime.
i s an
range.
function.
t h e r m a l l y accommodate o v e r a d i f f e r e n t
various
systems
t o p r o c e e d i n d e p e n d e n t l y o f t e m p e r a t u r e throughout
p r o p e r t y o f the
after
be u s e d i n
i s n o t so much t o make
t o group p r o c e s s e s a t the p h y s i o l o g i c a l
The
to
terms w h i c h w i l l
the r e a c t i o n s o f b i o l o g i c a l
s p e c i f i c m e a n i n g s t o terms w h i c h
that
i n body temperature,
function.
f o r the c o r r e c t n e s s o f c e r t a i n
literature
through-
d i s c u s s i n g a number o f t h e s e q u e s t i o n s i n d e t a i l ,
i s necessary to define
this
of
a t the b i o c h e m i c a l l e v e l ,
p a r t i c u l a r l y w i t h r e s p e c t t o enzyme
Before
a wide range
i n many c a s e s t o r e m a i n a c t i v e
l a r g e and o f t e n r a p i d changes
r a i s e s many i n t e r e s t i n g
it
to u t i l i z e
function
range
organism to a
new
time course o f thermal a c c l i m a t i o n f o r
functions
i n poikilotherms
i s generally i n
t h e o r d e r o f days o r w e e k s .
"Evolutionary adaptation" w i l l
which
o c c u r o v e r time
intervals
be
used to describe
l o n g e r than the l i f e
changes
span o f
2
the
i n d i v i d u a l organism.
c o v e r such changes
to
Thermal
as t h o s e u n d e r l y i n g
thermal environments
difference
i s greater
the a b i l i t y o f a
The
the
studies
general
able
questions.
i n this
answers t o the
a r e enzyme s y s t e m s
on the c a t a l y t i c
and
i n many
the maintenance
of function
following
poikilotherms
r a n g e s when t h e e f f e c t s
regulatory
many mammalian and b a c t e r i a l enzymes a p p e a r
with
thermal
t h e s i s were u n d e r t a k e n w i t h
to f u n c t i o n over wide temperature
temperature
temperature
compensated f o r by
at least partial
How
function
acclimation.
reported
hope o f p r o v i d i n g
would
inhabiting
i n c a s e s where t h e
t h a n c a n be
accommodation o r t h e r m a l
In
adaptation
proceed i n r e l a t e d stenothermic poikilotherms
different
of
evolutionary
properties
of
t o be i n c o m p a t i b l e
through such thermal
extremes?
p a r t i c u l a r , c a n s u c h phenomena as t h e r m a l accommodation,
acclimation
and
evolutionary
d i s p l a y e d by
poikilothermic
a t the
o f enzyme
level
The
problems
(i)
a d a p t a t i o n s to temperature
systems
o u t l i n e d as
Select a c r i t i c a l
interpreted
adopted
to i n v e s t i g a t e
(ii)
(iii)
p h y s i o l o g i c a l f u n c t i o n which i s
and
acclimation,
I s o l a t e a k e y enzyme f r o m t h i s
system
investigate
t h e e f f e c t s o f immediate
changes
thermal a c c l i m a t i o n
and
Compare t e m p e r a t u r e
with
these
follows:
known t o d i s p l a y b o t h t h e r m a l a c c o m m o d a t i o n
thermal
as
function?
experimental approach
c a n be
be o b s e r v e d a n d
thermal
and
temperature
upon t h e enzyme,
c h a r a c t e r i s t i c s of this
enzyme
s i m i l a r c h a r a c t e r i s t i c s o f h o m o l o g o u s enzymes
3
obtained
thermal
from s p e c i e s
s e l e c t e d as a s u i t a b l e
rationale behind this
of
2.
the
from the nervous
choice i s discussed
Accommodation and
Central
Nervous
probable
System
importance
poikilotherms
limiting
the o v e r a l l
1967;
Thermal
relationship
temperature
role
limits
f r o g s was
nervous
Nagai,
between c e n t r a l
tolerance
Brett,
1956;
1968).
The
nervous
the
system
1958;
following
i n establishing
a
the
o f the
Brecht found t h a t heat p a r a l y s i s i n
(1926) d e t e r m i n e d t h e u p p e r
system,
peripheral
thermal l i m i t s
i n s k a t e s a n d f l o u n d e r were a b l e
ephapses
Fisher,
and o c c u r r e d
nerve
c o n t r a c t i o n were i n h i b i t e d .
established
and
nervous
f u n c t i o n and
c o n f i n e d t o the c e n t r a l nervous
r e s p o n s e s and
of
tolerance,
(thermal accommodation range)
I n 1908,
c o n d u c t i o n and muscle
i n the
sytem
i n the c e n t r a l
temperatures below those a t which
across
sections
p r o c e s s have been c o v e r e d i n
( e . g . F r y , 1947;
P r o s s e r and
Acclimation
of thermal
o f changes
acclimation
whole organism.
and
The
i n the f o l l o w i n g
o b s e r v a t i o n s a r e p r o b a b l y the most r e l e v a n t
tissues
fish
of Poikilotherms
o f the c e n t r a l
i n setting
numerous r e v i e w s
at
of
enzyme f o r s u c h a s t u d y .
Thermal
Baslow,
sytem
introduction.
The
in
different
environments.
Acetylcholinesterase
was
inhabiting
that
functions
Battle
a t which
to e l i c i t
involving
various
physiological
synapses
s u c h a s t h e h e a r t pacemaker mechanism, c o n d u c t i o n
the nerve muscle
junction,
and
peristalsis
i n intenstinal
4
smooth m u s c l e ,
temperature
i t was
limit
failed
a t , o r s l i g h t l y below,
of the organism.
In a l a t e r
noted t h a t a t temperatures
reflexes
proposed
report
lethal
(Battle,
a p p r o a c h i n g the u p p e r
disappeared i n d e f i n i t e
t h a t death r e s u l t e d
the
sequence,
primarily
from
and
c o - o r d i n a t i n g mechanism.
Orr
similar
conclusion
i n Rana p i p i e n s .
coworkers
have demonstrated
physiological
in
f o r heat death
system
be most t e m p e r a t u r e
peripheral
Prosser,
nervous
1962;
sensitive,
f o l l o w e d by
function being least
P r o s s e r and
Farhi,
1965;
of
in fish
appears
thermal tolerance
The
acclimate
ability
t o be
a key
f o r the whole
o f the c e n t r a l
sensitive
factor
organism.
nervous
system
s t i m u l a t i o n of the r e t i n a
of
s e v e r a l weeks.
nervous
by
c e n t r a l response
Roots
a series
The
f u n c t i o n has
and
Prosser
low (4°C)
in fish
in
brain
rainbow
changes i n b o t h nerve
period
o f t h e r m a l a c c l i m a t i o n on
i n goldfish
central
a different
P r o s s e r and F a r h i
utilizing
they
conduction
t i m e o v e r an a c c l i m a t i o n
(1962) a n d
to
demonstrated
temperatures
a l s o b e e n shown i n q u i t e
of experiments
conditioned reflexes
effect
1968).
limits
By m o n i t o r i n g t h e m i d
were a b l e t o show c o m p e n s a t o r y
and
nervous
in setting
response
v e l o c i t y and
(Roots
of the c e n t r a l
(1964).
(16°C) a n d
to
P r o s s e r and N a g a i ,
b y K o n i s h i and H i c k m a n
t r o u t h e l d a t both high
appear
s p i n a l cord, with
t o t h e r m a l s t r e s s has been most c l e a r l y
to e l e c t r i c a l
neurosensitivity
Mid b r a i n f u n c t i o n s
Thus the t h e r m a l a c c o m m o d a t i o n r a n g e
system
P r o s s e r and
b y b o t h b e h a v i o u r a l and
of f i s h .
o f some
(1955) a r r i v e d a t a
techniques a h i e r a r c h y of temperature
the nervous
thermal
i t was
the f a i l u r e
central
192 9)
(1965).
the e s t a b l i s h m e n t of
they found t h a t both
the
way
In
5
lowest temperature a t which a c o n d i t i o n e d
established,
conditioned
w h i c h the
and
the
f i s h were
during
Baslow
temperature
response v a r i e d d i r e c t l y with
for
the
temperature
by
include
the
c h a n g e s i n s u c h f a c t o r s as
metabolites.
However, l i t t l e
Role of A c e t y l c h o l i n e s t e r a s e
i s known o f
(ACh)
as
a chemical
(1959), and
later
and
described
i n several recent
De
1969
1964).
of nerve
a c t s as
receptor
further
role
of
neuro-
The
by
to
the
been
(Nachmansohn 1967,
1968,
following outline of c h o l i n e r g i c
i s given by
i n the
Nachmansohn i n
n e r v e membrane
s i g n a l which
and
receptor
groups
f r o m the
i n the
conformational
the
following
i s recognized
protein located within
r e a c t i o n b e t w e e n ACh
carboxyl
Transmission
t r a n s m i t t e r a t the
reviews
of
function.
developments r e l a t i n g
impulses
is released
e x c i t a t i o n and
change
role
review.
ACh
specific
the
f u n c t i o n o f c h o l i n e r g i c systems have
Robertis,
transmission
his
and
j u n c t i o n h a v e b e e n s u m m a r i z e d i n a monograph
structure
1969;
i n Nerve
e a r l y s t u d i e s w h i c h e s t a b l i s h e d the
acetylcholine
enzyme
electrolyte distribution,
these changes i n thermal a c c l i m a t i o n of nervous
Nachmansohn
to
t h e r m a l a c c l i m a t i o n have been r e c e n t l y r e v i e w e d
various
muscular
the
in
structural lipids,
The
be
c e n t r a l nervous system
(1967) and
responses of
levels,
3.
could
acclimated.
Biochemical
fish
cold blocking
reflex
receptor
the
a
stereo-
membrane.
induces a
molecule,
releasing C a
protein.
The
free C a
c h a n g e s i n membrane
by
+ +
+ +
The
conformation
i o n s bound
ions
induce
phospholipids
to
6
and
other
polyelectrolytes, leading
permeability
across
the
and
the
movement o f
t o a change i n n e r v e membrane
f r o m 20,000 t o 40,000
membrane f o r e a c h m o l e c u l e o f ACh
initially
A c e t y l c h o l i n e s t e r a s e (AChE) r a p i d l y h y d r o l y s e s
the
receptor
p r o t e i n to r e t u r n
t h e r e b y r e - e s t a b l i s h i n g the
Nachmansohn p r o p o s e s t h a t
are
f u n c t i o n as
the
there
i s no
general
1959;
1969;
Changeux,
Podleski,
1969;
While
suggest
t h a t ACh
i t has
1966;
this
latter
Ehrenpreis,
of
1968;
1967;
point
essential
Karlin,
t h a t h y d r o l y s i s o f ACh
Importance of
Nervous
Durrell, et a l
this
by
1969).
has
action
(1969)
a t the
synapse
phospholipids.
AChE w o u l d r e m a i n
an
system.
C h o l i n e r g i c Mechanisms i n t h e
Central
System
In. s p i t e o f
a l a r g e volume o f
w i d e s p r e a d d i s t r i b u t i o n o f ACh,
(ChAc, the
1967;
Changeux e t a l , 1968,
a l t e r s membrane p e r m e a b i l i t y
component o f
(Nachmansohn,
cholinergic transmission
example,
that
However,
a l t e r n a t i v e mechanisms o f ACh
For
AChE
o f t e n been suggested
e n h a n c i n g e n z y m a t i c h y d r o l y s i s o f membrane
I t appears
4.
conformation
ACh-receptor molecule.
theory
have been p r o p o s e d .
its original
permitting
A C h - r e c e p t o r p r o t e i n and
a g r e e m e n t on
g a i n e d wide acceptance,
by
the
Hasson-Voloch,
this
ACh',
released.
membrane p e r m i a b i l i t y b a r r i e r .
s t r u c t u r a l l y l i n k e d and
AChE may
to
ions
enzyme
t h r o u g h o u t the
involved
vertebrate
c e n t r a l a c t i o n o f ACh
data r e l a t i n g
choline
i n synthesis
1945;
the
acetyltransferase
o f ACh)
and
c e n t r a l n e r v o u s s y s t e m and
(Feldberg,
to
Feldberg
and
AChE
to
the
Vogt,
1948;
7
B u r g e n and
Eccles,
Chipman,
1964;
Krnjevic,
of
b o u n d ACh,
e t a_l, 1967)
and
junction
(De
the
only
(Eccles
been w e l l
the
i s mediated
similar
that
central
proposal
reasons:
axonal
has
synapse
a central
Arnaiz
remains
the
cholinergic
s i g n i f i c a n t amounts
( S c h l a e p p e r and
(1959) has
the
of
far
Torack,
proposed that
at
the
neuromuscular
1966;
axonal
junction.
Consideration
been d i s c o u n t e d f o r the
i t i s not
possible
a p p l i c a t i o n of
the
to b l o c k at
blocking
t o x i c e f f e c t of
a p p l i c a t i o n o f ACh
a
Rodriguez DeLores
t h e n c h o l i n e r g i c mechanisms w o u l d be
these c o n d i t i o n s
specific
gradient
t h r o u g h a c h o l i n e r g i c mechanism e s s e n t i a l l y
conduction i s affected,
required
fractions
plasma membrane a t r e g i o n s
junctions
operating
generally
c o n d u c t i o n by
the
nervous
membrane
sucrose
documented t h a t
nervous t r a n s m i s s i o n .
1.
central
to
1969;
ChAC h a v e b e e n i s o l a t e d f r o m
1964;
Nachmansohn
conduction
i s true,
Koelle,
e t a_l, 1954) .
), and
to
synaptic
p r e p a r a t i o n s by
Robertis,
removed f r o m s y n a p t i c
1966
AChE and
1965;
1964;
c l e a r e v i d e n c e as
m o t o r neurone-Renshaw c e l l
AChE o c c u r w i t h i n
that
no
c l e a r demonstration of
I t has
for
Whittaker,
is still
cortex
centrifugation
this
1964;
A p r i s o n e_t a_l,
c h o l i n e r g i c mechanisms i n t h e
mammalian b r a i n
Brzin,
1963;
A l t h o u g h n e r v e e n d i n g and
containing
first
Hebb,
Robertis,
1969),, t h e r e
importance
system.
De
1951;
fails
general depolarization
of
neuromuscular
to b l o c k
axonal
i n h i b i t o r f a r exceeds
junction.
r e s u l t from a
compounds u s e d ;
t o e l i c i t an
possibly
this
p o t e n t AChE i n h i b i t o r s , o r i f
i s assumed t o
the
essential
following
i n many c a s e s
concentration
the
of
If
action
r e l a t i n g to
2.
Under
non-
direct
potential,
although
such f a c t o r s
as
8
altered
on
pH
or
i o n i c e n v i r o n m e n t may
occur.
Conclusions
s u c h r e s u l t s h a v e b e e n r e a d i l y d i s m i s s e d by
the
grounds t h a t
not
enough i s known a b o u t the
AChE and
the
ACh-receptor
reagents
(Nachmansohn,
evidence
i n d i c a t i n g that
p l a s m a membrane f r o m the
the
external
A r m e t t and
and
medium
Richie,
Rosenberg,
1968),
field
and
the
o u t e r membranes do
action
Deal,
1965;
concept of
gained
function
little
of
1957;
of
pharmacological
experimental
i n f a c t mask
o f AChE i n h i b i t o r s and
Rosenberg,
the
a x o n a l c o n d u c t i o n s has
this
the
on
availability
Despite considerable
(Walsh and
1960;
Nachmansohn
to e x t e r n a l l y a p p l i e d
1969).
based
Dettbarn,
Brzin,
ACh
the
in
1960a;b;
1966;
Martin
c h o l i n e r g i c a l l y mediated
support from workers
a x o n a l AChE r e m a i n s an
in
open
question.
The
release
mammalian c e r e b r a l
pathways and
cited
in
the
as
the
and
Chong,
has
suggested
not
suitable
brain
f o r the
Celesia
that
function
in cortical
activity
l e v e l s i n the
In r e c e n t
Russell
and
Jasper,
transmission
arousal,
or
central
Szerb,
1966).
s l o w and
but
nervous
afferent
also
1965;
Phillis
(1969)
diffuse release
may
play
a more
is
general
different
system.
investigators
These s t u d i e s ,
have
cholinergic
and
approach have been
Heller
been
mechanisms
r e l a t i o n s h i p s between c e n t r a l
W e i s s and
the
Krnjevic
i n maintaining
i n t h i s type of
(1969) and
of
cholinergic
( K a n a i and
animal behaviour.
problems i n v o l v e d
by
importance of
y e a r s a number o f
searched for possible
stimulation
in
r e t i c u l a r f o r m a t i o n has
t h i s type o f
for rapid
amounts o f ACh
following
nervous system
1965;
mechanisms and
substantial
cortex
mid
evidence
central
of
(1969).
In
the
reviewed
the
1950's
9
Rosenzweig
and
and coworkers
the e f f e c t s o f
"impoverished" b e h a v i o u r a l environments
AChE a c t i v i t y
was
to
investigated
i n the r a t c e n t r a l
nervous
such b e h a v i o u r a l
1957;
situations
B e n n e t t e t a_l, 1964) .
Russell
in
rats.
level
the
(see
established
AChE w i t h
organo-
1969).
Similar
(Glow a n d Rose,
responses
n o t l i n e a r , b u t below a
s p e e d o f e x t i n c t i o n was d i r e c t l y r e l a t e d
critical
activity,
t o AChE i n h i b i t i o n
e x p e r i m e n t s b y Glow a n d
1966; Glow e t a_l, 1966)
demonstrated
r e d u c t i o n o f AChE a c t i v i t y b e l o w 40 p e r c e n t o f t h e
normal v a l u e leads
While
t o a sudden
i t is difficult
increase
cholinergic
i n brain
ACh l e v e l s .
a t p r e s e n t t o e v a l u a t e the
importance o f such experiments
with
a dose-response
o f 40 t o 50 p e r c e n t i n h i b i t i o n o f n o r m a l AChE
Russell,
subjected
(Rosenzweig,
of learned behavioural
The r e l a t i o n s h i p was
coworkers
that
and the e x t i n c t i o n
and e v i d e n c e
i n animals
as maze t r a i n i n g
r e l a t i o n s h i p between i n h i b i t i o n o f b r a i n
phosphates,
upon t h e l e v e l s o f
system,
p r e s e n t e d f o r i n c r e a s e d AChE a c t i v i t i e s
"enriched"
i n terms o f u n d e r l y i n g
mechanisms, t h i s b e h a v i o u r a l a p p r o a c h , t o g e t h e r
the b i o c h e m i c a l and n e u r o p h y s i o l o g i c a l e v i d e n c e f o r the
presence and a c t i o n
system,
o f ACh a n d AChE i n t h e c e n t r a l
seems t o i n d i c a t e
mechanisms i n c e n t r a l
a definite role
nervous
for a suitable
outlined
for cholinergic
integration.
Thus AChE f r o m t h e f i s h
t o meet t h e c r i t e r i a
nervous
central
nervous
system
appears
a t the b e g i n n i n g o f t h i s
enzyme s y s t e m w i t h w h i c h
discussion
to study thermal
a c c l i m m a t i o n and e v o l u t i o n a r y a d a p t a t i o n t o temperature a t the
level
o f enzyme f u n c t i o n .
a critical
physiological
i t i s a n enzyme
process that
incorporated
i s known
into
to d i s p l a y
both thermal
10
a c c o m m o d a t i o n and
environmental
An
thermal a c c l i m a t i o n
i n response
to changing
temperature.
i n v e s t i g a t i o n i n t o the e f f e c t s o f immediate
c h a n g e s upon AChE a c t i v i t y
revealed
that
thermal
accommodation
of r e a c t i o n rate
does o c c u r a t p r o b a b l e p h y s i o l o g i c a l
concentrations.
The b a s i s
influence
of temperature
The
studied
these
phenomenon l i e s
upon enzyme s u b s t r a t e
e f f e c t s of thermal a c c l i m a t i o n
i n several
fish
for this
species
of t r o u t .
temperature
substrate
i n the
affinity.
upon AChE were
Following
acclimation
of
to d i f f e r e n t temperatures, a l t e r a t i o n s i n the
relative
proportions
analysis
o f t h e s e enzymes showed t h a t when t h e e n v i r o n m e n t a l
temperature
enzyme c o u l d
was
no
o f two
AChE v a r i a n t s were o b s e r v e d .
m a i n t a i n e d a t a l e v e l where one
longer
thermally
accommodate
o r where r e g u l a t i o n o f c a t a l y t i c
form o f the
for reaction
a c t i v i t y m i g h t be
second form
i s produced
f o r which
temperature
relationship i s better
Kinetic
lost,
t h e enzyme s u b s t r a t e
rate,
a
affinity-
s u i t e d f o r c o n t r o l of these
functions.
Comparisons
o f the p r o p e r t i e s
AChE enzymes f r o m d i f f e r e n t s p e c i e s
different
thermal environments
evolutionary
o f p r o b a b l y homologous
of fish
inhabiting
l e a d to the c o n c l u s i o n
a d a p t i o n o f AChE f u n c t i o n t o t e m p e r a t u r e
b a s e d upon s e l e c t i o n f o r an e n z y m e - s u b s t r a t e
relationship permitting
range
that
is
affinity-temperature
t h e r m a l accommodation o f
r a t e o v e r the temperature
markedly
reaction
n o r m a l l y e x p e r i e n c e d by
the
species.
I n answer t o t h e q u e s t i o n
these experiments,
i t c a n be
initially
stated
that
posed
in
designing
t h e r m a l accommodation,
t h e r m a l a c c l i m a t i o n and
evolutionary
as d i s p l a y e d b y many p o i k i l o t h e r m
and
i n t e r p r e t e d a t the
level
adaptation to
s y s t e m s c a n be
o f enzyme
function.
temperature
observed
12
METHODS
1.
Experimental Animals
A d u l t rainbow
trout
(Salmo
gairdnerii)
250 g were o b t a i n e d f r o m t h e Sun V a l l e y
B.C.
The f i s h were h e l d
c i r c u l a t i n g water
Feed'
gill
were u s e d
tolerate
Salt
i n P i n a s k Lake,
i n a number
temperatures
in
Lake C i t y ,
New
Utah).
i n the range
steel
o f 0° t o 2 5°C.
groups o f t r o u t
tanks i n which
not possible
acclimation
the temperature
Speckled trout
(Salvelinus
refrigeration
through
changed d a i l y .
The
these
natural
t a n k was m a i n t a i n e d t h r o u g h o u t t h e
p e r i o d o f f r o m 30 t o 36 d a y s ,
fed daily with Clark's
(generally
p o o l and p l a c e d
to c i r c u l a t e water
t a n k s , one q u a r t e r o f t h e volume was
photoperiod of the outdoor
trout
S_. g a i r d n e r i i c a n
t a k e n from the o u t d o o r h o l d i n g
As i t was
'Fish
Rainbow
c o u l d be c o n t r o l l e d a c c u r a t e l y w i t h h e a t i n g a n d
units.
Age
B.C. d u r i n g b o t h summer a n d w i n t e r
of experiments.
60 g a l l o n s t a i n l e s s
P o r t Moody,
outdoor tank w i t h
In a c c l i m a t i o n e x p e r i m e n t s ,
18 f i s h ) were
T r o u t Farm,
and f e d ad l i b on C l a r k ' s
( J . R. C l a r k Co.,
netted
i n a large
averaging about
'Fish
and the f i s h
were
Feed'.
(Salvelinus
fontinalis),
lake
namaychus) a n d t h e s p e c k l e d - l a k e h y b r i d ,
trout
splake,
were made a v a i l a b l e b y t h e U n i v e r s i t y o f T o r o n t o L a b o r a t o r y
for
E x p e r i m e n t a l Limnology,
Ontario.
acclimated
Immature
fish
Southern Research
Station,
(6-15 cm i n l e n g t h ) were
i n c i r c u l a t i n g water
Maple,
thermally
tanks a t the r e s e a r c h
station.
13
Trematonus b o r c h g r e v i n k i
Sound, A n t a r c t i c a d u r i n g
Brains
from
assayed
i n h a b i t e d by t h i s
in
the o r d e r
waters with
DeVries,
2.
The t e m p e r a t u r e
average
o f 0.1°C.
This
of the waters
-1.9°C, w i t h a n n u a l v a r i a t i o n s
species
has not been recorded
t h a n 2°C
Preparations
o f Rainbow T r o u t
d i s t i l l e d w a t e r and f r e e z e
solubilization
steps
Brain
Acetylcholinesterase
dried.
volume o f
The p r o c e d u r e f o r
o f t h e membrane b o u n d enzyme was b a s e d o n t h e
e x t r a c t i o n technique
described
by Morton
were c a r r i e d o u t i n a 4 ° C c o l d room.
b r a i n s were d i s p e r s e d
i n c o l d n-butanol
(1955). A l l
Freeze
dried
(1, g d r i e d t i s s u e t o
50 m l s o l v e n t )
by g r i n d i n g w i t h a m o r t a r and p e s t l e .
s u s p e n s i o n was
stirred
gravity
pellet
re-extracted
the
with
then c e n t r i f u g e d
-20°C.
acetone
n - b u t a n o l as b e f o r e .
After
The p e l l e t was
dispersed
and the
i n d r y a c e t o n e a t -20°C,
a t 10,000 x g r a v i t y f o r
d r i e d in. v a c u o o v e r c a l c i u m
removal o f the organic
d r i e d powder was
stirred
a t 10,000 x
discarded
t h e p e l l e t was
A f t e r complete
The
The s u p e r n a t a n t was
f o r 5 minutes and c e n t r i f u g e d
20 m i n u t e s .
at
f o r 2 hours,
f o r 15 m i n u t e s .
centrifuging
stirred
in
( s e e Somero a n d
P o o l e d b r a i n s were h o m o g e n i z e d i n a s m a l l
butanol
until
1967).
Preparation
cold
Somero.
d r i e d and s t o r e d a t -20°C
temperatures higher
Enzyme
(a)
1969.
fish
i n McMurdo
t h e summer o f 1965 b y D r . G. N.
6 f i s h were f r e e z e
i n November
were c a p t u r e d
chloride
solvents
t a k e n up i n c o l d 10
buffer,
pH 7.2,
gravity
f o r one h o u r t o remove i n s o l u b l e m a t e r i a l .
M
tris-HCl
f o r 2 hours and c e n t r i f u g e d a t 30,000x
The
s u p e r n a t a n t was
brought to
ammonium s u l p h a t e ,
at
and
the
to s e t t l e
supernatant adjusted
ammonium s u l p h a t e .
s e d i m e n t was
20
for at
stored
before
The
50
hour,
then
p e l l e t was
percent
solid
centrifuged
discarded
saturation with
10,000 x
i n d i s t i l l e d water.
i n 5 percent
l e a s t s i x months w i t h o u t
s o l u t i o n was
dialysed against
saturated
loss of
10 ^
gravity
This
preparation
ammonium
activity.
The
M tris-HCl buffer,
-
the
sulphate
enzyme
pH
7.2
Brain
and
washed
and
use.
Preparation
Spinal
of A c e t y l c h o l i n e s t e r a s e
Cord f o r
B r a i n and
s p i n a l c o r d were d i s s e c t e d o u t ,
volume o f
preparation
and
2,000 rpm
was
frozen
f o r 20
drawn o f f a n d
(c) E l e c t r i c
A
Eel
u s e d as
a bench c e n t r i f u g e .
a s o u r c e o f AChE f o r
purified
preparation
organ of e l e c t r i c
(Electric
V) .
an
T h i s m a t e r i a l had
(one^jL m o l a r u n i t w i l l
activity
on
8.0
The
The
at
supernatant
electrophoresis.
Acetylcholinesterase
Sigma C h e m i c a l Company
m i n u t e a t pH
c o l d d i s t i l l e d water.
thawed s i x t i m e s t h e n s p u n
m i n u t e s on
partially
f r o m the e l e c t r i c
from T r o u t
Electrophoresis
homogenized i n a s m a l l
was
f o r one
centrifugation at
t a k e n up
frozen
to
saturation with
After p r e c i p i t a t i o n overnight
c o l l e c t e d by
m i n u t e s and
c o u l d be
(b)
percent
10, 000 x g r a v i t y f o r 10 m i n u t e s .
solid
for
left
20
at
37°C) and
acrylamide
e e l was
disc
extracted
purchased from
eel Acetylcholinesterase
a c t i v i t y of
hydrolyze
o f AChE
l,00Cyu M o l a r
electrophoresis.
type
units/mg
1/AMole o f a c e t y l c h o l i n e
gave a s i n g l e b a n d o f
the
AChE
per
(d)
Preparation
of
Trematomus b o r c h g r e v i n k i
brain
Acetylcholinesterase
4 ml
of
u s e d as
3.
Freeze
dried brains
10
tris-HCl buffer,
M
- 2
a source
6 f i s h were h o m o g e n i z e d
pH
Assay of Acetylcholinesterase
the
acetylcholine
AChE a c t i v i t y
rate
can
of hydrogen
was
assayed
TTA
31)
gives
be
i n an
The
+ H^O
~ • ^ Choline
conveniently
a pH
volume o f
the
volume o f
In t h i s
titrator
stat with
against
rate
2 ml.
of a c e t y l c h o l i n e
buffer,
atmospheric
carbon dioxide
was
time
hydrolysis.
enzyme and
substrate
controlled
reaction vessel.
standardized
by
In a l l experiments
t o compensate f o r any
or
as
unit
(ACh)
type
uptake
non-enzymic h y d r o l y s i s o f
of
substrate.
Electrophoresis
Electrophoretic
by
M)
potassium b i p h t h a l a t e .
b l a n k s were r u n
Gel
- 2
activity
(Lauda B r i n k m a n , K-2/R)
s u r r o u n d i n g the
(generally 10
the
Copenhagen,
sodium h y d r o x i d e
T e m p e r a t u r e was
appropriate
4.
s t u d y , AChE
sodium h y d r o x i d e added per
coupled to a water j a c k e t
titration
following
(Radiometer,
a c c u r a t e l y with a c i r c u l a t i n g water bath
Sodium h y d r o x i d e
was
+ acetic acid
d e t e r m i n e d by
r e a c t i o n mixture contained
in a total
preparation
reaction
automatic
a measure o f
basic
This
Activity
ion production.
o p e r a t e d as
titrant.
7.2.
in
o f b r a i n AChE.
AChE c a t a l y s e s
The
from
standard
acrylamide
separation
of esterases
disc electrophoresis
was
(Davis,
carried
1964),
out
using
16
a
4 percent stacking gel, a 7 percent
5 x 10
M tris-glycine
- 3
applied
tank b u f f e r ,
s e p a r a t i n g g e l , and
pH 8.7.
Samples were
t o t h e t o p o f t h e s t a c k i n g g e l a n d r u n f o r 90 m i n u t e s a t 3
mA p e r tube
and a t 4°C.
E s t e r a s e a c t i v i t y was l o c a l i z e d w i t h i n t h e g e l b y t h e
«C n a p h t h y l
1959) .
4 x 10
acetate-diazonium
After
- 2
completion
salt
technique
then
and Hunter,
o f t h e r u n , g e l s were p l a c e d i n
M t r i s - H C l b u f f e r pH 7.1 f o r 10 m i n u t e s t o i n h i b i t
p r e c i p i t a t i o n o f t h e dye d u r i n g s t a i n i n g
and
(Market
transferred
to a solution
( A l l e n e t a_l, 1965)
containing«^naphthylacetate
(Sigma C h e m i c a l
Co., 40 mg/100 ml) a n d F a s t B l u e
R.R.
(Sigma C h e m i c a l
Co., 70 mg/100 ml) i n 4 x 1 0 ~
tris-HCl
buffer,
then
pH 7.1.
2
M
G e l s were r e a c t e d f o r 20 m i n u t e s a t 2 5°C
placed i n an a c i d - a l c o h o l s o l u t i o n
(ethanol:
.10 p e r c e n t
acetic
acid,
reduce
nonspecific staining.
water,
t h e g e l s c o u l d be s t o r e d i n d e f i n i t e l y a t 4 ° C .
5.
Protein
salt
3:2) f o r 30 m i n u t e s t o s t o p t h e r e a c t i o n a n d
After
rehydration i n d i s t i l l e d
Determinations
P r o t e i n c o n c e n t r a t i o n s o f AChE p r e p a r a t i o n s were
e s t i m a t e d b y t h e m e t h o d .of Lowry e t a__ (1951) .
diluted
to a concentration o f approximately
Samples were
20/u.g p r o t e i n / m l .
A s t a n d a r d c u r v e was p l o t t e d w i t h o v a l b u m i n i n t h e r a n g e
5 to
100/<A.g/ml f o r e a c h s e t o f d e t e r m i n a t i o n s .
6.
Sucrose
Gradient Centrifugation
Sucrose
g r a d i e n t s were p r e p a r e d
d u a l chamber g r a d i e n t maker.
i n 5 ml tubes w i t h a
Samples were r u n o n t h e S p i n c o
model L p r e p a r a t i v e u l t r a c e n t r i f u g e
equipped w i t h an SW
39
rotor.
For s t u d i e s o f the e f f e c t
sedimentation o f e l e c t r i c
g r a d i e n t s were p r e p a r e d
o f temperature
e e l AChE,
in 2 x 10
- 2
on t h e
5 t o 20 p e r c e n t
sucrose
M sodium b a r b i t o n e
buffer,
pH 7.2, a n d c o n t a i n e d 2 x 10 "'" M magnesium c h l o r i d e .
A
-
0.2 m l sample o f AChE d i s s o l v e d
chloride
and
s o l u t i o n was
i n t h e same b u f f e r - m a g n e s i u m
l a y e r e d onto
the t o p of each g r a d i e n t
r u n f o r s i x h o u r s a t 35,000 rpm a t t h e s p e c i f i e d
Drop c o u n t
f r a c t i o n s were c o l l e c t e d b y g r a v i t y a n d a s s a y e d f o r
AChE a c t i v i t y b y t h e s t a n d a r d method.
each
temperature
were c a l c u l a t e d
In the case
a c t i v i t y was
assay,
e t a l (1967).
readings.
d e t e c t e d i n s i t u b y t h e method o f J o l l e y
In t h i s
o f the r u n .
technique
sucrose
gradients are
s o l u t i o n and photopolymerized
G r a d i e n t s were f o r m e d b y
2.3 m l o f a 7.5 p e r c e n t a c r y l a m i d e
2 x 10 ^
refractometer
t o o low t o measure c o n v e n i e n t l y b y t h e s t a n d a r d
i n acrylamide
completion
from
Gradient densities a t
o f . t r o u t b r a i n AChE p r e p a r a t i o n s , where t h e
t h e enzyme was
prepared
temperature.
M magnesium c h l o r i d e
after
placing
gel solution containing
i n the d i s t a l
chamber o f t h e
g r a d i e n t maker a n d 2.5 ml o f t h e same s o l u t i o n c o n t a i n i n g
20 p e r c e n t
in
sucrose
i n t h e o u t l e t chamber.
0.2 ml o f t h e 7.5 p e r c e n t a c r y l a m i d e
applied
to the t o p o f the prepared
The sample,
gel solution,
dissolved
was
g r a d i e n t immediately
before
c e n t r i f u g i n g a n d r u n f o r t e n h o u r s a t 35,000 rpm a n d a t 4 ° C .
After
completion
of each
tube
o f t h e r u n , w a t e r was
to give a f l a t
under a f l u o r e s c e n t l i g h t .
l a y e r e d onto
the top
s u r f a c e , a n d the g e l p o l y m e r i z e d
The g e l s were t h e n
removed
from
18
the
c e n t r i f u g e t u b e s and
stained for esterase
described
previously for disc electrophoresis.
7.
Violet Difference
Ultra
a.
Introduction
ultra
violet
phenomenon has
region of
caused by
the
absorption
spectrum.
The
been reviewed by W e t l a u f e r
generally considered
is
that absorption
electron transitions
amino
the
230
r e g i o n r e s u l t s m a i n l y from e l e c t r o n
in
the
c a r b o x y l moeity of
studies,
shifts
i n absorption
terms of changes
the
1961;
i n the
p r o t e i n molecule
obtained
spectrum a t a reference
Bovey,
Thus, b y
absorption
transitions
I n a number
tertiary
1960;
plotting
temperatures
temperature
of
Foss,
absorption
to
induced
the
changes
can
spectra
i n a t w i n beam s p e c t r o p h o t o m e t e r i n w h i c h
the
s a m p l e s c a n be
reference
b.
obtained
read
relative
I d e a l l y , the
of
1960,
t h e UV
relative
thermally
detected.
structure
protein conformation
are
be
group.
s e c o n d a r y and
at different
i t is
s p e c t r a have been i n t e r p r e t e d
( Y a n a r i and
M a s s e y e_t a_l, 1966) .
spectra
tyrosine, whilst
peptide
this
acids
in
the
in
260-300rnytf.i n t e r v a l
tryptophan
my\A
and
(1962), and
i n the
i n the
patterns
basis of
phenylalanine,
in
as
Spectra
Proteins e x h i b i t c h a r a c t e r i s t i c
the
activity
to a c e l l h e l d at
difference
the
temperature.
Method
UV
s p e c t r a were d e t e r m i n e d w i t h
spectrophotometer equipped with
m e a s u r e m e n t s were made i n 1 cm
a C a r y 15
recording
a c i r c u l a t i n g water bath.
cells.
Electric
e e l AChE
All
in
was
dissolved in 2 x
10"^
M s o d i u m p h o s p h a t e b u f f e r , pH
t o a c o n c e n t r a t i o n o f 0.15
mg/ml, and
against
use.
this b u f f e r before
sample b u f f e r o n l y and
temperature.
was
extensively dialyzed
reference cuvette
maintained
S p e c t r a l scans
difference
spectrum f o r each
replotting
the
results
The
were r u n
a t the
to the
sample
in triplicate
t e m p e r a t u r e was
relative
7.2,
cuvette
and
obtained
spectrum
contained
by
obtained
a
RESULTS AND
1.
Partial
Purification
The
the
relative
of
Rainbow T r o u t
activities
e x t r a c t i o n procedure are
specific
a c t i v i t y of
about a four
homogenate,
fold
the
of
given
final
titration
plots
of
2 x
Characterization
a.
Central
lO '* M.
Although
represents
original
the
only
crude
down t o
an
activity
5 x
10 ^
-
could
M
not
non
be
ACh.
f r o m the
Rainbow
System
Introduction
(1957) as
and
throughout
W i t h c r u d e homogenates
-
Acetylcholinesterases
high
1.
of A c e t y l c h o l i n e s t e r a s e s
Nervous
Acetylcholinesterase
gave s t r a i g h t l i n e s
p l o t s were e n c o u n t e r e d and
Trout
i n Table
preparation
a c c u r a t e l y measured below about
2.
Brain
f r a c t i o n s taken
p u r i f i c a t i o n o v e r the
AChE c o n c e n t r a t i o n
linear
DISCUSSION
have been d e f i n e d
eserine-sensitive esterases
acetylcholine concentrations
which s p l i t
butyryl
which are
The
compound 284C51
Augustinsson
inhibited
( g e n e r a l l y 3 to
a c e t y l c h o l i n e a t a much h i g h e r
choline.
by
5 x
rate
10
by
M)
than
(Burroughs Wellcome)
at
- 6 - 5
concentrations
greater
to
10
M gives
a b o u t 100,000
i n h i b i t i o n o f AChE t h a n o f o t h e r
( A u s t i n and
b.
f r o m 10
Berry,
At
Central
least
cholinesterases
1953).
M u l t i p l e Forms o f A c e t y l c h o l i n e s t e r a s e
Trout
fold
Nervous
esterase
a c t i v i t y were
gel disc electrophoresis
r a i n b o w t r o u t b r a i n and
Rainbow
System
7 bands o f
following acrylamide
i n the
s p i n a l cord.
of
observed
extracts
S p e c i f i c AChE b a n d s
from
Table
1.
Partial
Brain.
P u r i f i c a t i o n o f AChE f r o m Rainbow
Trout
b
Fraction
Activity
B r a i n homogenate
Purification(X)
8.5
Recovery(%)
100
Butanol s o l u b i l i z e d
enzyme
11.9
1.4
107
20% ( N H )
fraction
23.7
2.8
53
32.3
3.8
25
4
SO.
2
20-50% (NH )„
S0
fraction
4
4
a.
b.
o
2 C acclimated
trout
Specific a c t i v i t y i s expressed
mg p r o t e i n / h o u r
as^M
ACh
hydrolysed/
were d e t e c t e d by t h e i r
i n h i b i t i o n with
10 ^ M e s e r i n e a n d w i t h
-5 . ~
10
M 284C51.
The AChE b a n d s o b t a i n e d w i t h
from rainbow t r o u t a c c l i m a t e d
are
both
the
shown i n F i g u r e
1.
spinal
t o 2 ° , 1 2 ° a n d 1 7 ° C f o r 32
Identical
cord preparations.
slower
f r o m warm a c c l i m a t e d
enzyme
estimated
present
r e s u l t s were o b t a i n e d
types.
AChE
migration
fish.
days
with
winter,
r a t e than
On t h e b a s i s o f s t a i n i n g
that equal
with
t o 12°C
possess
intensity
i t was
form
In f i s h
present;
captured
d u r i n g the
i n summer f i s h ,
the 17°C form i n e x c e s s .
h e l d a t 9°C i n the o u t d o o r p o o l
d u r i n g autumn a l s o h a d
enzymes, w i t h
the c o l d
c.
S p e c i f i c i t y and I n h i b i t i o n
Trout
does t h e enzyme
B r a i n e x t r a c t s from w i l d t r o u t
t h e 2 ° C enzyme was
enzymes u s u a l l y o c c u r r e d ,
Substrate
trout
amounts o f t h e two r a i n b o w enzymes were
i n the 12°C f i s h .
only
from c o l d a c c l i m a t e d
Trout acclimated
p o p u l a t i o n s were a l s o e x a m i n e d .
both
Fish
both
predominating.
Studies
o f Rainbow
Brain Acetylcholinesterases
Substrate
saturation plots f o r brain extracts
2 ° c a n d 17°C a c c l i m a t e d
and
preparations
p u r i f i e d e x t r a c t s a n d c r u d e homogenates o f b r a i n , a n d w i t h
shows a d i s t i n c t l y
both
brain
Figure
3.
rainbow t r o u t are g i v e n
from
i n Figure
B o t h enzymes show g r e a t e r a c t i v i t y w i t h
2
ACh
than w i t h
p r o p i o n y l - c h o l i n e o r b u t y r y l c h o l i n e , and i n each
substrate
i n h i b i t i o n occurs
a t concentrations
case
above a b o u t
_o
3 x 10
M ACh.
preparations
The r e l a t i v e
and e l e c t r i c
s u b s t r a t e s and i n h i b i t o r s
activities
e e l AChE w i t h
are given
o f rainbow t r o u t
individual
i n Table
2.
No
and p a i r e d
summation
23
Figure
1.
Resolution
o f Rainbow T r o u t
Acrylamide Gel Disc
Electrophoresis
B r a i n AChEs b y
Electrophoresis.
conditions:
a t 3 mA
a n d 400 v o l t s
glycine
tank b u f f e r ,
90
per g e l .
pH
8.7
2°C a c c l i m a t e d
trout
17°C a c c l i m a t e d
trout
12°C a c c l i m a t e d
trout
minutes
Tris-
24
Figure
2.
Substrate S p e c i f i c i t y o f AChE from 2 ° C
A c c l i m a t e d Rainbow T r o u t ,
in 10~
2
M tris-HCl
-2
10
Standard assay
b u f f e r , pH 7.2 w i t h
.
.
M sodium h y d r o x i d e as t i t r a n t .
temperature 2 ° C .
©
Acetylcholine
iodide
_
Propionylcholine
A
Butyrylcholine
iodide
iodide
Assay
25
Figure
3.
Substrate
Acclimated
S p e c i f i c i t y o f AChE from
Rainbow T r o u t .
as i n F i g u r e
2.
17°C
Standard a s s a y
Assay temperature
©
A c e t y l c h o l i n e -iodide
E2
Propionylcholine
A
Butyrylcholine
iodide
iodide
15°C.
Table
2.
Surnmation E x p e r i m e n t s w i t h
Effect
Choline
o f I n h i b i t o r s on H y d r o l y s i s
T r o u t and E l e c t r i c
Esters
and the
o f ACh b y Rainbow
E e l AChEs
R e a c t i o n r a t e (JK M s u b s t r a t e
hydrolysed/mg
protein/hr)
Substrate
(2.5 x 10~ M)
2 C trout
AChE
3
17 C t r o u t
AChE
electric eel
AChE
(xl0~ )
3
Acetylcholine
54.0
43.2
71.0
9.2
7.3
61.0
Propionylcholine
Butyrylcholine
0
A c e t y l c h o l i n e and
propionylcholine
A c e t y l c h o l i n e and
butyrylcholine
Propionylcholine
butyrylcholine
22.1
11.9
4.3
15.9
64.0
5.3
9.9
3.5
8.0
and
0
A c e t y l c h o l i n e and
5 x 10~ M eserine
0
3
0
A c e t y l c h o l i n e and
1 0 " M 284C51
0
6
A s s a y s were c a r r i e d
0
o u t a t 10 C f o r t h e r a i n b o w
a n d a t 2 5°C f o r t h e e l e c t r i c
Substrates:
0
Acetylcholine
butyrylcholine
t r o u t enzymes
e e l AChE
iodide;
iodide.
propionylcholine
iodide;
27
of
activity
occurred
w i t h any s u b s t r a t e
that butyryl or propionyl
cholinesterases
to the r a t e o f h y d r o l y s i s .
either
pair tested,
Hydrolysis
indicating
are not c o n t r i b u t i n g
o f 2.5 x 1 0
M ACh b y
- 3
t h e 2 ° . a n d 1 7 ° C t r o u t AChE o r t h e e e l enzyme,
5 x 10
-5
completely
i n h i b i t e d with
M eserine,
M 284C51.
These r e s u l t s o b t a i n e d
inhibitors
i n d i c a t e t h a t e s s e n t i a l l y a l l o f the
with
a c t i v i t y o f the t r o u t b r a i n e x t r a c t s
and w i t h
specific
detected
was
-6
10
substrates
and
esterase
by the a s s a y
method c a n be a t t r i b u t e d t o AChE.
d.
E f f e c t o f pH o n A c e t y l c h o l i n e s t e r a s e
The
bell
shaped p H - a c t i v i t y curves f o r the rainbow
t r o u t AChEs shown i n F i g u r e
4 a r e s i m i l a r t o those
w i t h AChEs f r o m a v a r i e t y o f s o u r c e s
Bull
e.
Activity
and L i n d q u i s t ,
( B e r n s o h n e t a_l, 1963;
1968; S i l m a n a n d K a r l i n ,
Sucrose Gradient
obtained
1967).
C e n t r i f u g a t i o n o f Rainbow T r o u t
Brain
Acetylcholinesterases
The
brain
r e s u l t s o f the experiments
preparations
up i n a c r y l a m i d e
were c e n t r i f u g e d
detected,
as
fish.
on s u c r o s e g r a d i e n t s
g e l s o l u t i o n a r e shown i n F i g u r e
r e s u l t s were o b t a i n e d
acclimated
i n which rainbow
with extracts
In each case
5.
made
Identical
from b o t h 2° and 17°C
3 bands o f e s t e r a s e
activity
t h e c e n t r a l b a n d s e d i m e n t i n g i n t h e same p o s i t i o n
electric
e e l AChE.
Inhibition with
10~
5
M 284C51 a n d
-4
10
trout
M eserine
d i d n o t remove a n y one band, b u t g r e a t l y
reduced the i n t e n s i t y o f the c e n t r a l band.
were
28
Figure
4.
Influence
o f pH on the A c t i v i t y o f Rainbow
T r o u t B r a i n AChEs.
Sodium phosphate
buffer,
-2
10
M, was
used i n the range pH 6-8, and
-2
-2
t r i s - H C l b u f f e r , 10
M, c o n t a i n i n g 10
M
sodium c h l o r i d e from pH 7.5 t o
©
9.5.
17°C a c c l i m a t e d rainbow
Assay temperature
trout.
17°C.
o
E3
2 C a c c l i m a t e d rainbow
Assay temperature
2°C.
trout.
Rate (uM ACh hydrolyzed / m g protein /hr)
29
Figure
5.
Sucrose Gradient
Trout
C e n t r i f u g a t i o n o f Rainbow
and E l e c t r i c
20 p e r c e n t
7.5 p e r c e n t
2 x 10
layered
- 1
sucrose
E e l AChEs.
gradient
acrylamide
M MgC-L_.
on t o p .
A 5 ml 0 t o
was
formed i n
gel solution
containing
Samples o f 0.2 ml were
Centrifugation
conditions:
10 h o u r s a t 100,000 x g r a v i t y a t 4 ° C .
centrifugation
and
stained
t h e g e l s were
f o r esterase
After
photopolymerized
activity.
electric eel
AChE
2 ° C acclimated
frout
17°C acclimated
trout
2° or 17°C
ated trout.
acclimStained
in the presence
T0 /M
_6
284C51
of
30
The
following conclusions
of
these r e s u l t s .
in
3 molecular
1.
Rainbow t r o u t b r a i n e s t e r a s e s
s i z e c l a s s e s , AChE p r o b a b l y s h a r i n g
w i t h a t l e a s t one o t h e r
esterase.
warm a n d c o l d a c c l i m a t e d
s i m i l a r molecular weight,
3.
2.
t r o u t and from e l e c t r i c
a b o u t 260,000
both
e e lare of
(Leuzinger
from
amount o f m a t e r i a l
to characterize
e t a_l, 1 9 6 9 ) .
Trematomus
from the f o l l o w i n g r e s u l t s t h a t
available i t
t h i s enzyme s y s t e m a s f u l l y
as was done f o r t h e r a i n b o w t r o u t A C h E ' s .
by
a class
Bra-in
Because o f the s m a l l
not possible
occur
The AChEs f r o m
Characterization of Acetylcholinesterase
borchgrevinki
was
c a n be made o n t h e b a s i s
the t o t a l
I t was c o n c l u d e d
activity
t h e a s s a y c o u l d be a t t r i b u t e d t o a s i n g l e AChE
detected
species.
—6
1.
The p r e s e n c e o f 2 x 10
M 284C51 i n t h e a s s a y
c o m p l e t e l y i n h i b i t e d ACh h y d r o l y s i s .
2.
Acrylamide
gel electrophoresis
of the crude
homogenate gave f o u r b a n d s o f e s t e r a s e
brain
activity;
only
— ft
one
o f t h e s e b a n d s was i n h i b i t e d b y 5 x 10
and
10
M 284C51
-4
4.
M eserme.
E f f e c t o f A s s a y T e m p e r a t u r e upon t h e K i n e t i c s o f A c e t y l c h o l i n e
Hydrolysis
a.
by Acetylcholinesterase
E f f e c t o f T e m p e r a t u r e o n t h e Maximum V e l o c i t y o f
Acetylcholine
Hydrolysis
Numerous a t t e m p t s h a v e b e e n made t o e s t a b l i s h
r e l a t i o n s h i p s between the thermal
and
temperature
tolerances
of
poikilotherms
d e p e n d e n t c h a r a c t e r i s t i c s o f t h e i r enzymes.
The
31
c h a r a c t e r i s t i c s most commonly i n v e s t i g a t e d a r e
f o r o p t i m a l enzyme a c t i v i t y a t V max
enzyme t h e r m o s t a b i l i t y .
recentlyby L i c h t
others.
(1967),
I n most c a s e s b o t h
temperatures
levels of substrate,
Studies of this
Read
the
type have been
(1967), a n d
Ushakov
thermal optima
and
and
reviewed
(1967) among
thermal
d e n a t u r a t i o n occur a t temperatures
above t h o s e
in
i n t e r e s t with reference to
the
the e n v i r o n m e n t .
Of p a r t i c u l a r
present study are r e s u l t s
Kusakina
(1963) h a s
the
temperature
for
a t which
of muscle
thermal environment
50
a positive
muscle
of cottoid
fish.
a c t i v i t y was
muscle
Similarly,
e x c i t a b i l i t y ceased.
(1964) f o u n d no d i f f e r e n c e
habitat
that
temperature
certain
required
and
f o r the organism,
n o t be
Kfigrelli
Alexandrov
of proteins
is clearly
to
not
thermal
setting
t h e r m o s t a b i l i t y per se, b u t r a t h e r ,
i n turn a l t e r
to
a
molecule
a c t i v i t y and
Changes i n t h e r m a l l y d e p e n d e n t
(1969)
between
the p r o p e r t y s u b j e c t
f o r s u c h f u n c t i o n s as c a t a l y t i c
f l e x i b i l i t y may
which
cholinesterase
c o n f o r m a t i o n a l f l e x i b i l i t y o f the p r o t e i n
regulation.
which
incubation
above t h o s e a t
killifish.
the r e s i s t a n c e
the
temperature,
Baslow and
b u t where t h e r m o s t a b i l i t y
limits
s e l e c t i o n may
after
and
at
i n c a s e s where a c o r r e l a t i o n e x i s t s
temperature
denaturation,
lost
i n the l e v e l s o f
a c t i v i t y o f c o n t r o l and h e a t k i l l e d
i s lost,
temperature
increased with increasing habitat
12° t o 15°C
proposed
relationship
ihactivation,
The
and o c c u r r e d a t t e m p e r a t u r e s
has
cholinesterases.
excitability
cholinesterase
percent of cholinesterase
30 m i n u t e s
obtained for f i s h
demonstrated
between the temperature
encountered
allosteric
conformational
the t h e r m a l d e n a t u r a t i o n c h a r a c t e r -
istics
o f the molecule.
c o n f o r m a t i o n a l changes
detail
i n following
Criticism
Certain aspects of thermally
i n protein structure
sections of this
against
dependent
are discussed i n
thesis.
the use o f t h e r m a l optima
f o r maximum
enzyme a c t i v i t y a n d t h e r m o s t a b i l i t y a s m e a s u r e s o f t h e r m a l
adaptation i s generally
of
l e v e l e d a t the non-physiological
the e x p e r i m e n t a l methods.
a t which
enzyme a c t i v i t y
F o r example,
the a c t u a l
nature
temperature
i s m a x i m a l depends o n s u c h f a c t o r s a s
a s s a y t i m e , a n d t h e V max l e v e l s o f s u b s t r a t e e m p l o y e d a r e
g e n e r a l l y f a r i n excess o f probable p h y s i o l o g i c a l substrate
concentrations.
Similarly,
dependent
upon s u c h f a c t o r s
pH,
environment,
ionic
and o t h e r c e l l u l a r
Bowen a n d K e r w i n ,
protein thermostability
as presence or.absence
i s highly
of substrate,
a n d b i n d i n g o f t h e enzyme t o membranes
structures
( V e s s e l l a n d Y i e l d i n g , 1966,
1956; Cheeseman e_t a_l,
c o r r e l a t i o n between iri v i t r o
1967) .
Thus a n y
enzyme t h e r m a l o p t i m a o r
t h e r m o s t a b i l i t y w i t h environmental temperature,
may be s i m p l y
fortuitous.
Arrhenius
rainbow
are
from
t r o u t a c c l i m a t e d t o 2° and 17°C and f o r e l e c t r i c e e l
shown i n F i g u r e 6.
the r e a c t i o n
of
p l o t s o f l o g V o p t . v e r s u s 1/T f o r AChEs
the f i s h .
i n each
c a s e t h e maximum v e l o c i t y o f
i n c r e a s e s w i t h temperature
beyond the thermal
range
W i t h b r a i n AChE f r o m Trematomus b o r c h g r e v i n k i
t h e a c t i v i t y o f t h e enzyme a t h i g h l e v e l s
o f s u b s t r a t e (10
M
ACh)
i s t w i c e as g r e a t a t 10°C as i t i s a t 2°C (see T a b l e 5 ) .
This
f i s h has n o t been r e c o r d e d i n waters
optimal
temperature
o f enzyme s t r u c t u r e ,
f o r enzyme a c t i v i t y ,
above 2 ° C .
Thus, t h e
and thermal
stability
are probably not important factors i n
33
F i g u r e 6.
A r r h e n i u s P l o t s o f AChE A c t i v i t y f o r the
Rainbow Trout and E l e c t r i c E e l Enzymes.
-2
Standard assay i n 10
M tris-HCl buffer,
pH 7.2 w i t h V opt. l e v e l s o f ACh.
o
©
17 C a c c l i m a t e d t r o u t AChE
13 2°C a c c l i m a t e d t r o u t AChE
A
e l e c t r i c e e l AChE
.do^
6o|
34
setting
thermal
I t has
adapted
Although
temperature
Brown,
1968)
has
thermal energy
a l o w e r i n g o f the energy
been demonstrated
1963;
Kwon and
Olcott,
1965;
no means u n i v e r s a l
the
be
(Ea).
environmental
1964;
(Vroman
Hochachka,
Hochachka
and
19 6 8 ) .
which decrease
t r o u t and
as
the t e m p e r a t u r e
( W i l s o n and
Cabib,
v a r i e t y of sources
interpreted
1956),
and
(Chadwick,
the decrease
reaction.
straight
They proposed
line
through
1957).
temperature
temperature
range
where t h e o t h e r s t e p i s s l o w e r .
and
are comparable.
the r a t e
energy
of a c t i v a t i o n .
versus
1/T
and
vary with
plots
range
a different
j o i n e d by a c u r v e d p o r t i o n over
as
limiting
curved
e e l AChE
from
Cabib
a
(1956)
for electric
s t e p f o r the
eel
overall
t h a t A r r h e n i u s p l o t s would give a
i s rate
temperatures
Similarly
W i l s o n and
steps
rates
eel
activation
f o r an e l e c t r i c
i n the r a t e
the
limiting,
energies of
for cholinesterases
i n Ea w i t h
AChE i n t e r m s o f a change
electric
i s raised.
p l o t s have been 'reported p r e v i o u s l y
Km
of a c t i v a t i o n
Somero and
(Read,
in cold
i s low may
i n a number o f c a s e s
AChEs t h e c u r v e d A r r h e n i u s p l o t s y i e l d
both
species.
r a t e s o f enzyme a c t i v i t y
I n t h e c a s e o f the r a i n b o w
be
i n these
a c o r r e l a t i o n b e t w e e n Ea a n d
i t i s by
Somero,
that
where a v a i l a b l e
through
such
f o r AChE a c t i v i t y
been argued
organisms
maintained
and
limits
The
straight
These two
the t h e r m a l
It i s also
of
line
range
need not g i v e s t r a i g h t
that
would
where
at higher
the s t e p which had
suggested
log V
the
lower
max
l i n e s when b o t h V
(see b e l o w ) .
the
i n the
lines
curve would f l a t t e n
approached
temperature
where one
max
Non-linear Arrhenius
plots
o b t a i n e d w i t h amino o x i d a s e h a v e b e e n r e l a t e d
temperature
dependent
conformations
to a
t r a n s i t i o n o f t h e enzyme b e t w e e n
( K o s t e r and V e e g e r ,
1968;
M a s s e y e t a_l,
Recently,
e v i d e n c e has been p r e s e n t e d f o r s i m i l a r
dependent
t r a n s i t i o n s between m u l t i p l e
esterases
and AChE f r o m e r y t h r o c y t e s
forms
from
1966).
temperature-
o f serum
(Main,
cholin-
1969).
E n e r g i e s o f a c t i v a t i o n f o r the t r o u t
e e l AChEs were c a l c u l a t e d
two
and
the r e l a t i o n s h i p
electric
l o g (K
- K
)=
—*
E
a
4.6
(-^— 1^
absolute
-ji—) where K a n d
T-/
1
temperatures
activation.
drawing
The
2
n
are r e a c t i o n
and T_
and Ea
Ea v a l u e s a t a s p e c i f i c
velocities at
i s the energy
t e m p e r a t u r e was
a t a n g e n t t o the A r r h e n i u s curve a t t h a t
relationships
b e t w e e n Ea a n d
temperature
of
o b t a i n e d by
temperature.
are given i n Table
3.
For
given
temperature
difficult
value
the rainbow
enzyme a t 2°C.
electric
(
2 5°C)
The v a l u e o f 1.9
concluded that
E f f e c t o f Temperature
effectors
are
as
the
K c a l / m o l e o b t a i n e d f o r the
temperature
o f the t r o u t
enzymes a t
2°C.
no c l e a r r e l a t i o n s h i p e x i s t s
between
habitat
studied.
on E n z y m e - S u b s t r a t e
C u r r e n t m o d e l s o f enzyme r e g u l a t i o n
of
results
t h a n the v a l u e of the 2°C
a p p a r e n t e n e r g i e s o f a c t i v a t i o n o f AChE and
i n the s p e c i e s
any
o f a d a p t i v e advantage,
17°C enzyme i s l o w e r a t 17°C
i s lower than e i t h e r
temperature
b.
i n terms
The
e e l enzyme a t t h e p r o b a b l e h a b i t a t
I t must be
the
enzymes t h e Ea v a l u e s a t
a r e e s s e n t i a l l y t h e same.
to i n t e r p r e t
f o r the
trout
i n m o d i f y i n g enzyme-substrate
Affinity
stress
the
affinities
importance
(Atkinson,
36
Table
3.
Apparent Energies
T r o u t and E l e c t r i c
AChE s o u r c e
of Activation
E e l AChEs a t s e v e r a l
(°C)
2°C a c c l i m a t e d t r o u t
(Ea) f o r t h e Rainbow-
E
a
temperatures
(^cal/mole)
2
3.6
17
1.8
2
4.1
17
2.1
25
1.9
17
3.0
o
17 C a c c l i m a t e d
Electric
eel
trout
1966;
Stadtman,
1966).
I t has been s u g g e s t e d r e c e n t l y
t h e c a s e o f enzymes from p o i k i l o t h e r m s ,
a role
analogous
by a l t e r i n g
to that of p o s i t i v e
enzyme-substrate
b r i n g about compensatory
(Hochachka
and
The
and
of
Somero,
i n s u c h a way
as
of the
enzyme,
7 and
(as m e a s u r e d b y
c o n s t a n t , Km)
of rainbow
the
and a s s a y
reciprocal
temperature,
t h e r m a l range
o f t h e enzymes f o r the ACh
and approach maximal v a l u e s
c o r r e s p o n d i n g to those a t which
acclimated.
This
case o c c u r s a t about
25°C,
at
a temperature
temperature
A
temperature
has been o b s e r v e d f o r p y r u v a t e k i n a s e s from rainbow
1968)
for lactate
Trematomus
(Hochachka
and Hochachka,
and
dehydrogenases
1969),
and
and
trout,
1968)
dehydrogenases
lungfish
fructose
trout
(Somero a n d
tuna,
and
Hochachka,
lungfish,
and k i n g - c r a b
f o r glucose-6-phsophate
6-phospho-gluconate
1969) , f o r s a l m o n
Somero,
from
i n this
similar
and e n v i r o n m e n t a l
Trematomus b e r n a c c h i i
and
corresponding closely
temperature.
c o r r e s p o n d e n c e b e t w e e n minimum Km
fish
were
e e l enzyme; the minimum Km
t o the p r o b a b l e minimum h a b i t a t
the a n t a r c t i c
each
vary with
the f i s h
r e l a t i o n s h i p between h a b i t a t
a l s o h o l d s f o r the e l e c t r i c
that
of
(minimum Km)
temperatures
Km
trout
8.
p a r t o f the b i o l o g i c a l
the a f f i n i t i e s
temperature
reactions
organism
I n t h e c a s e o f t h e t r o u t AChEs i t i s a p p a r e n t
the upper
to
1968).
the a p p a r e n t M i c h a e l i s
over
play
i n t h e r a t e s o f enzyme
t h e r m a l range
e e l AChE f o r ACh
shown i n F i g u r e s
may
negative effectors
r e l a t i o n s h i p s b e t w e e n the a f f i n i t y
electric
are
and
affinities
changes
throughout the b i o l o g i c a l
temperature
that i n
(Somero
dehydrogenase
f r o m k i n g - c r a b (Somero,
diphosphatases
(Behrisch,
38
Figure
7.
E f f e c t of Assay Temperature on the Km o f
ACh f o r AChEs from
17° and 2°C A c c l i m a t e d
-2
Rainbow T r o u t .
tris-HCl buffer,
Standard assay xn 10
pH 7.2.
M
The enzymes were
assayed at.ACh c o n c e n t r a t i o n s i n the range
4
3
10
to 5 x 10
M and Km v a l u e s were
determined
(1/V versus
from d o u b l e - r e c i p r o c a l p l o t s
1/ ACh ).
@
17°C a c c l i m a t e d t r o u t AChE
El
2°C a c c l i m a t e d t r o u t AChE
-
Temperature (°C)
39
Figure
8.
Effect
o f Assay
Temperature
ACh f o r E l e c t r i c
in
10~
2
M tris-HCl buffer,
enzyme was
in
E e l AChE.
Standard
pH 7.2.
assay
The
a s s a y e d a t ACh c o n c e n t r a t i o n s
the range
10
-4
t o 5 x 10
v a l u e s were d e t e r m i n e d
plots
o n t h e Km o f
(1/V v e r s u s
from
1/ ACh ) .
-3
M a n d Km
double-reciprocal
5
01
0
l
I
i
1
1
1
1
l
5
10
15
20
25
30
35
40
Temperature (°C)
1969;
B e h r i s c h and Hochachka,
isocitrate
citrate
dehydrogenases
synthases
At
physiological
is
clear.
increase
f o r rainbow
and Lewis,
above t h a t a t w h i c h
significance
i n temperature
would
t h e v e l o c i t y o f t h e enzyme r e a c t i o n ,
of
temperature.
characteristic
reaction
This
rate
remains
relationship
be e x p e c t e d t o
this
relatively
o f b o t h forms
o f t r o u t AChE.
The 'warm' f o r m
the
f o r m shows t h e r e l a t i o n s h i p above a b o u t
i s reflected
substrate
independent
type o f temperature-independence i s
r e l a t i o n s h i p a t temperatures
effect
effect i s
affinity.
shows t h i s
'cold'
and
Km i s minimum, t h e
o f t h e Km-temperature
Although a r i s e
the o v e r a l l
NADP
1970).
counteracted by a decrease i n enzyme-substrate
Hence,
trout
(Moon, p e r s o n a l c o m m u n i c a t i o n )
(Hochachka
temperatures
1969),
i n the r a t e s
above a b o u t 1 7 ° C ;
2°C.
This
of acetylcholine hydrolysis a t
c o n c e n t r a t i o n s a p p r o a c h i n g t h e minimum Km
(Table 4 ) .
-4
In t h i s
c o n n e c t i o n , a r e c e n t e s t i m a t e o f 2.16 x 10
c o n c e n t r a t i o n o f ACh r e l e a s e d
into
the s y n a p t i c
M f o r the
space
o f the
v e r t e b r a t e motor e n d p l a t e c o r r e s p o n d s c l o s e l y
t o t h e minimum
Km v a l u e o f b o t h
1969).
effects
t r o u t AChEs
(Namba a n d Grob,
A considerable
overcompensation
o f temperature
upon r e a c t i o n
_o
f o r the a c c e l e r a t i n g
rate
i s observed with the
o
2 C t r o u t enzyme a t 18 C, a n d w i t h t h e e l e c t r i c
40°C
(Table 4 ) .
physiological
However,
this
importance.
e f f e c t may be o f l i t t l e
In the case o f the 2°C t r o u t
the decrease i n r e a c t i o n r a t e
i n v i v o by the appearance
e e l AChE a t
i s presumably
enzyme
compensated f o r
o f t h e 1 7 ° C enzyme, w h i l e t h e e l e c t r i c
e e l may n o t e n c o u n t e r w a t e r
temperatures
as h i g h as 40°C.
41
Table
4.
Rates
for
o f ACh H y d r o l y s i s a t Minimum
Km
AChEs f r o m Rainbow T r o u t , E l e c t r i c
Levels
o f ACh
E e l and
Trematomus
AChE
source
ACh
2°C
acclimated
trout
2.5 x
17°C
acclimated
trout
(M)
10~
2.5 x 10
Electric
eel
10
Temperature
a t minimum
Km (°C)
4
4
17
25
4
-4
Trematomus
borchgrevinki
a.
Rate
1.5 x 10
of hydrolysis
o f minimum
b.
The r a t e
to
V/V
(minimum
Km)
a
0
0.79
5
1.10
8
1.10
12
0.90
18
0.61
11
0.61
13
0.80
22
1.00
27
1.20
15
0.42
20
0.92
30
1.10
35
1.10
40
0.65
10
0.51
a t assay temperature/rate a t temperature
Km
o f ACh h y d r o l y s i s
the r a t e
Assay.
temperature
(°C)
a t 2°C
a t 10°C i s e x p r e s s e d
relative
At
lower thermal
relationship
i s reversed.
enzyme t h e Km r i s e s
15°C,
reaching
t h e Km-temperature
F o r t h e 'warm' f o r m o f t h e t r o u t
s h a r p l y as the temperature
a value
minimum v a l u e .
so h i g h
extremes
falls
a t 10°C t h a t i s about 4 times the
Thus, a t 2 ° C t h e Km o f t h e enzyme i s p r o b a b l y
a s t o make t h e enzyme e s s e n t i a l l y
inactive
a t low a n d
presumably p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s .
a
fall
i n temperature
increase
from 20°C t o 15°C l e a d s
i n t h e Km o f t h e e l e c t r i c
enzyme-substrate
rates
affinity
i s reflected
This
Trematomus b o r c h g r e v i n k i ,
a v a i l a b l e was s u f f i c i e n t
temperatures.
i n a r e d u c t i o n o f the
(Table 4 ) .
t h e amount o f enzyme
o n l y f o r Km d e t e r m i n a t i o n s
Lineweaver-Burk p l o t s o b t a i n e d
t h e c r u d e b r a i n homogenate a r e p r e s e n t e d
effect
several
o f assay
temperature
substrate
on Km,
concentrations,
the t r o u t and e l e c t r i c
r e a c t i o n rates given
i n Figure
9.
The
and on r e a c t i o n r a t e s a t
are given
e e l AChEs,
i n Table
a t two
a t 2°C and 10°C
i n Table
the a f f i n i t y
Trematomus enzyme f o r ACh c h a n g e s m a r k e d l y w i t h
The
decrease i n
o f ACh h y d r o l y s i s a t low s u b s t r a t e c o n c e n t r a t i o n a t
For
with
Similarly,
to a 3 f o l d
e e l enzyme.
t e m p e r a t u r e s b e l o w t h e minimum Km p o i n t
with
below
5.
As
o f the
temperature.
5 show t h a t a t low s u b s t r a t e
-4
concentrations
(below 5 x 10
overcompensation
ACh
and
forreaction rate.
i s considerable
F o r example, a t 1.5 x 1 0
t h e r a t e o f ACh h y d r o l y s i s a t 1 0 ° C i s o n l y
rate
as
M ACh) t h e r e
a t 2°C.
Again,
this; e f f e c t
Trematomus b o r c h g r e v i n k i
i s of l i t t l e
52 p e r c e n t
importance
- 4
o f the
i n vivo
does n o t i n h a b i t w a t e r s above 2 ° C ,
d i e s w i t h i n 81 m i n u t e s a t 1 0 ° C (Somero a n d D e V r i e s ,
1967).
M
43
F i g u r e 9.
E f f e c t o f Assay Temperature
on the Km o f ACh
f o r Trematomus b o r c h g r e v i n k i AChE.
Lineweaver-
Burk P l o t s a t 2°C (M ) and 10°C ( © ) .
Standard assay i n 1 0
pH 7 . 2 .
- 2
M tris-HCl buffer,
Table
5.
Effect
o f Temperature
Hydrolysis
u p o n t h e Km a n d Rate o f ACh
f o r B r a i n AChE f r o m
Trematomus
borchgrevinki
Reaction rate
^nM ACh h y d r o l y s e d / m l / h r )
Parameter
ACh
10"
10°C
46
91
49
50
39
26*
(M)
3
5 x 10"
4
2.5 x 1 0 "
1.5 x 10
Km
2°C
4
-4
31
a
1.5 x 1 0 ~
a.
a
16
d
4 x
4
Values obtained by e x t r a p o l a t i o n
Burk p l o t s .
from
10~
Lineweaver-
3
45
Clearly,
more d a t a must be
Km-temperature
such
relationships
a remarkably
The
rate,
o b t a i n e d b e f o r e the
c a n be
a s s e s s e d f o r AChE
expressed
as Q--,
over
o f minimum Km
temperature
Trematomus AChEs a r e g i v e n i n T a b l e
and
electric
e e l AChEs a 2 t o 3 f o l d
approximately
Main
kinetic
from
Conformation
(1969) has
6.
above
the
electric
F o r the
i n Km
over
10°C
hydrolysis
value.
Changes i n Km
solubilized
presented evidence
observed with
such
enzyme.
indicating
e r y t h r o c y t e AChE a r e
I f t h e Km-temperature
the s o l u b i l i z e d
f i s h AChEs a r e
aggregation i t i s d i f f i c u l t
effect
could occur
t o see how
that
exists
that
artifact
o f the
the e f f e c t
electric
by
t h e Km-temperature
iri v i t r o
of temperature
e e l AChE o v e r
temperature
was
c e n t r i f u g a t i o n and
assay
Thus,
relationship
system.
relationships
Km-temperature
the range
i n v e s t i g a t e d by
ultraviolet
possibility
To examine
where Km
sucrose
difference
structural
i s simply
upon t h e m o l e c u l a r
of
d e p e n d e n t upon
the
the
the
dependent
in v i v o where t h e enzyme f o r m s a
component o f t h e n e u r a l membrane.
and
Eel Acetylcholinesterase
upon t h e r m a l l y i n d u c e d c h a n g e s i n m o l e c u l a r a g g r e g a t i o n
the
eel
trout
t h e minimum Km
Induced
of E l e c t r i c
p r o p e r t i e s of bovine
reaction
c o n s t a n t r a t e o f ACh
R e l a t i o n s h i p between T h e r m a l l y
Structural
ranges
change
at substrate concentrations approaching
c.
and
f o r the rainbow t r o u t ,
and
m a i n t a i n an
of
stenothermal species.
r e l a t i o n s h i p s b e t w e e n change i n Km
temperature
will
significance
an
this
question,
conformation
i s markedly
of
affected
gradient
spectroscopy.
46
Table
6.
Relationship
b e t w e e n Km Change a n d Q
o f ACh H y d r o l y s i s
Approaching
Trout,
AChE s o u r c e
2°c
a t C o n c e n t r a t i o n s o f ACh
t h e Minimum Km
Electric
ACh
f o r AChEs f r o m
E e l a n d Trematomus
(M)
Rainbow
borchgrevinki
Temperature
range(C)
Q
Km
10
acclimated
trout
17°C
2.5 x 1 0
2-12
0.9
2.2
2.5 x 10
17-27
1.2
2.4
10
25 - 35
1.1
3.0
2 - 10
0.4
26.8
- 4
acclimated
.
trout
Electric
eel
Trematomus
borchcrrevinki
- 4
1.5 x 1 0 "
Q-Lo v a l u e s were c a l c u l a t e d
Q_0
are
o f t h e Rate
1 Q
reaction
=
( l/ 2)
rates
v
v
l
0
4
from
/
/
t
l
the r e l a t i o n s h i p
t
2
a t temperatures
where V
t-^ a n d t
2
1
and
V
2
respectively
47
AChE f r o m
electricus
has
a molecular weight
subunit structure
S t u d i e s by
that
the e l e c t r i c organ
o f the
Changeux
ionic
increasing at ionic
MgClj.
Sucrose
15°,
2 5° a n d
10
G r a f i u s e t a l . (19 68)
ionic
t h e Km
environment
this
1966)
t h e c e n t r i f u g a t i o n medium a t 15
Lineweaver-Burk p l o t s
in
t h e Km
In
o f t h e enzyme w i t h
sucrose
coefficients
Km
in this
sample a n d
protein
, 25
u
and
indicate
33
C.
sample.
under these
The
However, s i n c e the
expected
type
sedimentation
by
running
method c o u l d n o t
of both
the
the
(see M a r t i n a n d Ames, 1961), a n d
the
solution
over
be
test
t o change w i t h
to the v i s c o s i t y o f
o f the s u c r o s e
change
conditions.
sedimentation c o e f f i c i e n t
i s directly proportional
f u n c t i o n of temperature
The
a significant
sedimentation behaviour
c e n t r i f u g a t i o n medium
viscosity
o
n
temperature
t h e s t a n d a r d m i g h t be
temperature.
a
s t u d y as
the
d e t e r m i n a t i o n s were made
of p r o t e i n s are g e n e r a l l y determined
the t e s t
some
d e p e n d e n t upon
gradient studies of this
known s t a n d a r d s w i t h
used
i n F i g u r e 11
component
(Figure 10).
o f AChE i s known t o be
(Changeux,
%
of
t o t a l a c t i v i t y , with
O
in
10
patterns obtained at
show a s i n g l e m a j o r
fraction
shown
the
33°C f o r e l e c t r i c e e l AChE i n t h e p r e s e n c e
f o r the m a j o r i t y o f the
a
AChEs i s
p o l y d i s p e r s i t y of
gradient centrifugation
p o l y d i s p e r s i t y about
As
of e l e c t r i c organ
have
s t r e n g t h s below t h a t o f 2 x
M magnesium c h l o r i d e
accounting
possess
( L e u z i n g e r e_t . a l , 1969) .
environment,
system
2 x
and
the s e d i m e n t a t i o n b e h a v i o u r
d e p e n d e n t upon t h e
o f 260,000 a n d
2tl2(± t y p e
(19 66)
o f the e e l E l e c t r o p h o r u s
is essentially
the temperature
a
range
of
linear
investigated,
48
F i g u r e 10.
Sucrose G r a d i e n t Sedimentation
of E l e c t r i c E e l AChE a t 15°C
25° C ( © )
and 33°C
(A).
Profiles
( • ),.
Experimental
c o n d i t i o n s are g i v e n i n the methods
section.
relative
A c t i v i t i e s are expressed
to the f r a c t i o n w i t h the
h i g h e s t a c t i v i t y i n each
experiment.
I
1
1
1
I
-
I
0
5
10
15
20
25
30
Fraction Number
TOP
E f f e c t o f Temperature on the Km o f ACh
f o r E l e c t r i c E e l AChE Assayed i n the
C e n t r i f u g a t i o n Medium.' Linweaver-Burk
P l o t s a t 15°C ( I I ) , 25°C
33°C ( A ) .
(©)
and
i t was
argued
protein
of
that
i f the s e d i m e n t a t i o n c o e f f i c i e n t
does n o t change s i g n i f i c a n t l y w i t h
sedimentation, distance versus
line
temperature,
or
conditions.
Marked changes i n p r o t e i n c o n f o r m a t i o n
alter
f o r a standard set of
the s e d i m e n t a t i o n b e h a v i o u r
the s e d i m e n t a t i o n c o e f f i c i e n t
temperature,
w o u l d be
w o u l d be
One
o f t h e enzyme and,
changed i n a l i n e a r
The
plot
large
to conclude
enough t o be
(b) c h a n g e s may
that either,
d e t e c t e d by
temperature
over a temperature
be
accompanied by
attributed
l a r g e Km
drawn f r o m
Figure
fashion with
structural
a way
range
13. A l t h o u g h
that
spectra with changing
protein denaturation.
change
the
sedimentation
where t h e Km
temperature
Thus, a l t h o u g h
the
Km
s m a l l c o n f o r m a t i o n a l changes i n
of subunits.
difference
to those
25°C
cannot
i n molecular
S i m i l a r c o n c l u s i o n s can
spectra plotted
i n t h e enzyme o c c u r
temperature,
are s m a l l i n comparison
linear.
fashion with
changes presumably r e s u l t i n g
conformational alterations
(as a
occurred or,
t o s u c h m a j o r c h a n g e s as a l t e r a t i o n s
the u l t r a v i o l e t
temperature
f o r the
d i f f e r e n c e b e t w e e n 15° a n d
aggregation or d i s s o c i a t i o n
be
unless
is clearly
in a linear
r e l a t i o n s h i p shows a s h a r p c h a n g e .
t h e enzyme, t h e
(a) no
have o c c u r r e d i n such
o f t h e enzyme a l t e r e d
be
and
t h e method h a s
coefficient
c h a n g e s may
to
of sedimentation distance
e e l AChE i s shown i n F i g u r e 12,
is left
or
expected
f u n c t i o n o f f r a c t i o n number) a g a i n s t t e m p e r a t u r e
electric
experimental
plots of sedimentation distance against
non-linear.
plots
viscosity,
a straight
temperature
the
temperature,
should y i e l d
molecular aggregation with
of
in
from
i n the
ultraviolet
the a c t u a l absorbance
accompanying such
events
changes
as
51
Figure
12.
Effect
o f T e m p e r a t u r e upon t h e
Behaviour
o f E l e c t r i c E e l AChE.
represents
The
error
Sedimentation
t h e mean o f t h r e e
bars
indicate
Each p o i n t
determinations.
the range about
mean, a n d a r e i n t h e o r d e r o f - 0.5
(approximately
t
0.08 m l ) .
number i s e x p r e s s e d
fraction
in
number t o compensate
each group o f experiments
Experimental
fractions
fraction
as a f u n c t i o n o f t o t a l
t h e number o f f r a c t i o n s
methods
The
for variation
collected i n
(27 t o 32
fractions).
c o n d i t i o n s are g i v e n i n the
section.
this
in
_l
K
o
suoipojj
I
o
JO * O N
o
1 —
o
p+oi
/0|S|
UO|pDJj
52
Figure
13.
Ultraviolet
Eel
Difference Spectra
AChE as a F u n c t i o n o f
Spectra
are p l o t t e d
o b t a i n e d a t 2 5°C.
of E l e c t r i c
Temperature.
relative
t o the
spectrum
Wavelength (mu)
53
5.
Thermal
Accorniaodation, T h e r m a l A c c l i m a t i o n ,
A d a p t a t i o n t o Temperature
the N e r v o u s
a.
Thermal
System
physiological
range
relationships
electric
at
for Acetylcholinesterase
u n a f f e c t e d by
appears
temperature
t o l i e i n the
shown i n F i g u r e
temperatures
14.
approximately constant rate
o f ACh
t h e Trematomus enzyme i n s u f f i c i e n t
accommodation and
occur over temperature
The
reaction
ranges
i s s e t by
temperatures below t h i s
change
hydrolysis
level
of
10°C
m a i n t a i n an
6).
d a t a were a v a i l a b l e
thermally
With
to
i n d u c e d Km
changes
the a n i m a l .
f o r t h e r m a l accommodation o f
a t t h e minimum Km.
affinity
falls
and
At
rapidly
the
thermal
the r e a c t i o n
rate at
low
concentrations i s maintained at a r e l a t i v e l y constant
to temperatures beyond
i s the
limiting
the organism,
n o t an
over
and
decrease.
( a b o u t 2 5°C) o f t h e a n i m a l .
system
trout
(Table
the r e a c t i o n r a t e
I n t h e t r o u t AChE s y s t e m
substrate
i n Km
point w i l l
( T a b l e 4) as b o t h t h e e n z y m e - s u b s t r a t e
energy o f the system
Km-temperature
the temperature
point
may
particular
n o r m a l l y e n c o u n t e r e d by
minimum t e m p e r a t u r e
rate
over a
F o r the rainbow
above the minimum Km
i f rate
t h a t a t low and probably-
c o n c e n t r a t i o n s t h e enzyme r e a c t i o n
e e l AChE's, a 2 t o 3 f o l d
determine
from
Fish
f o r the o b s e r v a t i o n
substrate
relatively
temperature
Evolutionary
Accommodation
The b a s i s
remain
of
and
system
the u p p e r
Thus,
nervous
the temperature
hydrolysis
in setting
tolerance
i f the c e n t r a l
in setting
t h e r a t e o f ACh
important f a c t o r
thermal
the upper
range
b y AChE i s p r o b a b l y
thermal
limit.
54
Figure
14.
E f f e c t of Assay Temperature
on the Km o f
ACh f o r AChEs from Rainbow Trout,
Eel
and Trematomus b o r c h g r e v i n k i .
E3
2°C a c c l i m a t e d
trout
©
17°C a c c l i m a t e d
trout
A
electric eel
O
Trematomus b o r c h g r e v i n k i
Electric
Temperature (°C)
55
It
other
i s possible,
than r e a c t i o n
Km c h a n g e s ,
thermal
however, t h a t
rate
and t h a t
are a f f e c t e d by the t h e r m a l l y
these
factors
fluctuations
activity.
i n t h i s respect
i n substrate
saturation
plots
2 5 C, t h e t e m p e r a t u r e
fluctuations
value
(0.98 x 1 0 ~ ) c a n g r e a t l y
achieve
the
a f f e c t the rate
point,
value,
Km
o f enzyme
switch.
As
25°C,
are required
to
A s i m i l a r s i t u a t i o n i s observed with
Trematomus a n d r a i n b o w t r o u t AChEs
Km
15.
a b o u t t h i s minimum
c h a n g e s i n ACh c o n c e n t r a t i o n
t h e same e f f e c t .
minimum
substrate
a t t e m p e r a t u r e s above a n d b e l o w
Thus, a s t h e Km o f t h e enzyme
the
i n the
e s s e n t i a l l y as an a l l - o r - n o t h i n g
increases
much g r e a t e r
i n d u c e d Km c h a n g e s u p o n t h i s
i n AChE c o n c e n t r a t i o n
4
Km v a l u e
small
enzyme
e e l AChE shown i n F i g u r e
small
acting
of
a t w h i c h t h e Km r e a c h e s a minimum
0
activity,
One m e c h a n i s m
to regulate
i s demonstrated
for electric
induced
i n setting
i s the a b i l i t y
concentration
The e f f e c t o f t h e r m a l l y
f o r m o f enzyme r e g u l a t i o n
the
are involved
l i m i t s f o r t h e enzyme s y s t e m i n v i v o .
w h i c h may be i m p o r t a n t
At
enzyme c h a r a c t e r i s t i c s
increases
(Figures
16 a n d 1 7 ) .
a t temperatures
above
t h e a d v a n t a g e s t o be o b t a i n e d f r o m
rate
s t a b i l i z a t i o n may be o f f s e t b y l o s s o f c o n t r o l o v e r t h e
reaction.
Km
i s accompanied by a decrease
control
in
A t t e m p e r a t u r e s b e l o w t h e minimum
over the r e a c t i o n .
t h e AChE s y s t e m ,
investigators
tolerance
i n both r e a c t i o n
t h e n b o t h enzyme r e g u l a t i o n
enzyme s y s t e m j_n v i v o .
have a t t e m p t e d
or organisms w i t h
point,
rate
i f t h i s form o f c o n t r o l
a c c o m m o d a t i o n may be i m p o r t a n t
the
Km
increasing
and
i s important
and r a t e
i n s e t t i n g thermal
limits for
As d i s c u s s e d
previously,
to c o r r e l a t e
the upper
the t h e r m o s t a b i l i t i e s
many
thermal
o f enzymes
56
F i g u r e 15.
ACh S a t u r a t i o n Curves o f E l e c t r i c E e l
ACh a t 15°
40°C ( A ) .
(<§), 25° ( B ) , and
Relative
Activity
57
F i g u r e 16.
ACh S a t u r a t i o n Curves of Trematomus
b o r c h g r e v i n k i AChE a t 2° ( H ) and
10°C
( ® ).
100
ACh]
(mM)
58
Figure
17.
ACh
S a t u r a t i o n Curves of
Rainbow T r o u t
2°
( ©
),
12°
AChE a t 0°
( A
) and
2°C
acclimated
( EH ) ,
18°C
( O
).
80
59
from p o s s i b l y
limiting
physiological
relationships
o b s e r v e d w i t h t h e f i s h AChE's p r o v i d e an
a l t e r n a t e mechanism b y w h i c h
inactivated
i n vivo
b.
Acclimation
Thermal
(i) Adjustment
systems.
enzymes may
i n the absence
be
The
thermally
of protein denaturation.
o f the t h e r m a l accommodation
range
I n t h e t r o u t b r a i n AChE s y s t e m a d j u s t m e n t
accommodation range
by
regulating
in
response
proposed
a
following
the r e l a t i v e
a second
(2° a n d
The
17°C)
where t h e r e l a t i v e
two
and
i s produced
i s better
suited
has
The
and Hochachka,
1968),
Somero,
isocitrate
lactate
c o m m u n e i a t i o n ) and c i t r a t e
to
longer thermally
catalytic
f o r which
the
for control
p r o d u c t i o n o f enzyme
a l s o been o b s e r v e d w i t h t r o u t
1970).
is altered
of
together a t i n t e r m e d i a t e temperatures
c u r v e f o l l o w i n g changes
1968),
It is
amounts o f e a c h enzyme v a r i e s w i t h
temperature.
types
enzymes o c c u r s i n g l y a t t h e r m a l
w i t h a l t e r e d and a p p a r e n t l y a d a p t i v e changes
temperature
enzyme
o r where r e g u l a t i o n o f
form
relationship
functions.
acclimation
rate,
thermal
i s achieved
p r o p o r t i o n s o f t h e two
f o r m o f t h e enzyme c a n no
i s lost,
Km-temperature
extremes
thermal a c c l i m a t i o n
t h a t when t h e e n v i r o n m e n t a l t e m p e r a t u r e
accommodate f o r r e a c t i o n
these
o f the
to changing environmental temperature.
r a n g e where one
activity
Km-temperature
Km-
i n environmental
dehydrogenases
dehydrogenases
synthases
variants
i n the
pyruvate kinases
the
temperature
(Somero
(Hochachka
and
(Moon, p e r s o n a l
(Hochachka
and
Lewis,
60
(ii)
Rate c o m p e n s a t i o n o f AChE
An
i n t e r e s t i n g feature
enzymes i s t h a t
activity
o f t h e two r a i n b o w t r o u t AChE
t h e minimum Km v a l u e s a r e s i m i l a r a l t h o u g h
occur a t widely d i f f e r e n t temperatures.
presumably p h y s i o l o g i c a l substrate
catalysed
rate
Thus,
than the r e a c t i o n
catalysed
a t low a n d
concentrations
b y t h e ' c o l d ' enzyme a t 2 ° C w i l l
they
the r e a c t i o n
proceed a t a slower
b y a n e q u a l amount o f t h e
'warm' enzyme a t 1 7 ° C i f t h e two enzymes h a v e s i m i l a r
turnover
numbers.
T h e r e a r e a number o f f a c t o r s w h i c h may a c t t o r a i s e t h e
rate
o f ACh h y d r o l y s i s
i n the c o l d a c c l i m a t e d
+
e t a l (19 64)
in
o b s e r v e d changes i n b r a i n
o
r a i n b o w t r o u t t r a n s f e r r e d f r o m 16
effects
(Table
ionic
7) shows t h a t a t h i g h
strength
leads
been r e p o r t e d
marmorata
the
is
+
Na , K
o
and CI
t o 6 C.
ACh c o n c e n t r a t i o n s
t o a marked i n c r e a s e
rate
1966).
o f the r e a c t i o n
increased.
Thus,
the rate
increasing
A similar relationship
organ o f Torpedo
However, a t l o w e r ACh
generally
thermal a c c l i m a t i o n
An
increase
decreases as i o n i c
o f ACh h y d r o l y s i s
i n intracellular
temperature
been observed
i n several
1969).
concentrations
strength
i n v i v o may be
occur
during
process.
environmental
Wilson,
t o 2°C
i n b o t h Km a n d
f o r AChE f r o m t h e e l e c t r i c
(Changeux,
levels
A study o f the
modulated by changes i n the i o n i c environment t h a t
the
Hickman
o f s a l t s on b r a i n AChE f r o m t r o u t a c c l i m a t e d
maximum v e l o c i t y o f ACh h y d r o l y s i s .
has
state.
and b l o o d
pH o n l o w e r i n g
( a b o u t 0.014 pH u n i t s
poikolotherms
(Rahn,
p e r °C) has
1965; Reeves a n d
W i t h t h e 2 ° C t r o u t AChE, a f a l l
i n temperature
61
Table
7.
Effect
o f S a l t s on t h e Km
a n d Rate o f
o f ACh
b y AChE f r o m 2 ° C A c c l i m a t e d Rainbow T r o u t
relative
Salt
10" M
NaCl
3
5 x
Km
x
10"
Hydrolysis
activity
3
4
ACh
(M)
2.5
x 10"
4
5 x
10"
4
saturating
3.3
0.36
0.58
0.96
4.1
0.35
0.58
1.06
10" M
3
NaCl
10~ M
NaCl
7.7
0.31
0.61
1.50
10" M
KC1
4.9
0.35
0.61
1.20
4.4
0.45
0.70
1.33
MgCl^
7.0
0.32
0.62
1.42
Control
2.7
0.44
0.63
1.00
2
2
2 x
10~ M
MgCl
5 x
10~ M
3
2
3
S t a n d a r d a s s a y i n 10""
2°C.
2
a.
M tris-HCl
buffer,
A c t i v i t i e s are expressed r e l a t i v e
control
a t s a t u r a t i n g ACh
levels
pH
7.2
(control)
at
t o the a c t i v i t y o f the
62
f r o m 17°
ACh
t o 2°C
hydrolysis
(Figure
4)
and
t h r o u g h an
Nigrelli
total
cold acclimation
state,
as
trout acclimated
or
of
the
p r o p o s e d by
Baslow
compensation of
brain
17°C
f o r 35
days
killifish.
(Table
8)
failed
to
the
i t can
be
argued that
same r a t e o f ACh
hydrolysis
not
in
b e c a u s e o f a l t e r e d ACh
of neural
membranes w h i c h may
A l t h o u g h no
levels in fish,
significant
i t may
information
a number o f
changes
be
the
concentrations,
affect
is available
on
investigators
i n membrane s t r u c t u r e
following
acclimation.
unsaturation
acclimation
1968)
phopholipids,
of
the
Roots
total
temperature
this
same
percent
membranes
of
(1964) f o u n d a t r e n d
i n the
gold
t r e n d was
choline
These two
the
towards
brain f a t t y acids with
particularly
glycerophosphotides.
60
specific
cold acclimated.states
J o h n s t o n and
over
enzyme
a c t i v i t y o f AChE : f r o m
c h o l i n e r g i c mechanisms.
(Roots,
amount o f
might
the
changes i n p r o p e r t i e s
thermal
compensation
acclimated
hand,
to m a i n t a i n
have r e p o r t e d
activity
difference.
other
a l t e r e d b r a i n ACh
of
i n thermally
t o 2 ° and
significant
warm and
that rate
i n the
activity
and
rate
substrate.
increase
However, e s t i m a t i o n s
On
possible
(1964) t o a c c o u n t f o r r a t e
cholinesterase
necessary
i n the
r e l a t i o n s h i p b e t w e e n pH
considered
i n the
show any
increase
holds at p h y s i o l o g i c a l l e v e l s of
achieved
present
r e s u l t i n an
i f the
I t was
be
could
total
and
and
decreasing
in a later
established
f o r the
study
brain
ethanolamine
phospholipid
lipid
(Cotman e t a l , 1 9 6 9 ) .
fish,
increased
types account
i n rat brain synaptic
It i s generally
agreed
for
plasma
that
63
Table
8.
Specific
Activities
Acclimated
o f B r a i n AChE f r o m Rainbow T r o u t
t o 2 ° a n d 17°C f o r 35
days
Specific
Acclimation
t e m p e r a t u r e ( C)
Number o f
f i s h assayed
A
s
s
a
y
activity
temperature
18
3
(°C)
10
2
2
12
10.1
± 0.1
8.6
± 0.3
6.2
± 0.3
17
11
10.1
± 0.3
8.4
± 0.1
6.2
± 0.1
Whole b r a i n homogenates
(100 mg b r a i n / m l ) were a s s a y e d i n
10~
pH 7.2, w i t h 2 x 1 0 ~
2
M tris-HCl buffer,
3
M ACh a s
substrate.
a.
Specific
p e r mg
the
activities
a r e e x p r e s s e d asyu-M ACh h y d r o l y z e d
p r o t e i n per hour.
mean.
± v a l u e s i n d i c a t e the range
about
the
lipid
role
c o n s t i t u e n t s o f b i o l o g i c a l membranes p l a y a
i n r e g u l a t i o n of
for
t h i s has
and
Harland
permeability,
b e e n somewhat i n d i r e c t .
(1969) have
phospholipid
Cl~
ionic
content
Tobias
o f f r o g s k i n s and
e_t a l , 1962), and
Thus,
there
species
f r o m the
(Vanatta,
i s considerable
1969;
support
t h a t c h a n g e s i n the
lipid
during
t h e r m a l a c c l i m a t i o n may
membranes
b i n d i n g of
f o r the
necessary
be
induced
compensated
maintenance a t
(Fast,
to
f o r the
composition
f o r by
the
the
lipid
species
a l t e r e d temperature
stabilization
of
the
similar
t o t h a t f o l l o w e d by
present.
1967;
various
1968).
by
Roots
membranes
nerve
i n t i s s u e s from
reflect
t h a t can
not
Continued
g e n e r a l l y leads
i o n i c balance
and
+
maintenance
underlying
c h a n g e s i n membrane p e r m e a b i l i t y
gradual
et
ionic
Na
similar
suggestion
e x p o s e d t o r a p i d c h a n g e s i n t e m p e r a t u r e may
thermally
the
Papahadjopoulos,
o f membrane p e r m e a b i l i t y c h a r a c t e r i s t i c s
fish
the
component o f n e u r a l
be
A l t e r a t i o n i n the
ions
evidence
Watlington
properties of
artificial
(19 68)
conduction.
example,
o t h e r workers have r e a c h e d
from s t u d i e s w i t h
phospholipid
For
most
found a c o r r e l a t i o n between
t r a n s p o r t systems w h i l e
conclusions
although
critical
over a time
changes i n t i s s u e l i p i d s
to
a
course
(Hickman
a l . 1964) .
It
free
should
be
stressed that phospholipids
i n n e u r a l membranes, b u t
possibly
i n c o r p o r a t i n g AChE.
when c o n s i d e r i n g t h e
AChE, as
i t has
enzyme a c t i v i t y
effects
been found
can
be
r a t h e r as
T h i s may
of
do
lipoprotein
be
o f some
not
occur
complexes
importance
t h e r m a l a c c l i m a t i o n upon
i n other
a l t e r e d by
membrane s y s t e m s
removal of
the
that
phospholipid
component.
For
example,
phospholipds are
respiratory activity
i n mitochondria,
and
requirement f o r such
f o r m an
enzymes as
absolute
.cytochrome o x i d a s e
f_-hydroxy-butyric
cytidine
diphosphocholine
1965), and
transferase
Thus t h e
lipids
following
possibility
of b r a i n
attributed
One
W i t h the
to the
point
f r o m warm and
lipid
that
should
need f o r r a t e
the
r e l a t i o n s h i p b e t w e e n in
concentrations.
rate
activity
not
concentration
It
state
w i t h any
Strickland,
comparisons
butanol-acetone
fish
that could
overlooked i n
failed
be
present
excess i n
i n enzyme
possible
activity
to
less e f f i c i e n t
s u c h d i s c r e t e s y s t e m s as
little
maintain
levels with a
large
enzyme.
t o d e t e r m i n e enzyme and
degree of a c c u r a c y .
the
temperature might have
still
is
substrate
i n great
above c r i t i c a l
of c a t a l y t i c a l l y
considering
thermal a c c l i m a t i o n
then a r e d u c t i o n
hydrolysis
within
free
v i v o enzyme and
I f AChE was
is difficult
concentrations
be
compensation d u r i n g
e f f e c t i f i t was
o f ACh
lipid
cold acclimated
f o l l o w i n g a drop i n environmental
the
(Tanaka and
component.
the
physiological
and
Schneider,
r a i n b o w t r o u t AChEs,
changes i n s p e c i f i c
warm a c c l i m a t e d
1965) ,
e x i s t s t h a t c h a n g e s i n membrane
made b e t w e e n c r u d e homogenates and
t o show any
MacLennan,
t h e r m a l a c c l i m a t i o n m i g h t h a v e some e f f e c t
upon AChE a c t i v i t y .
extracts
and
( F i s c u s and
ATPase
1962)
mitochondrial
(Sekuzu e t a l , 1963)
f o r membrane bound Na-K
1965).
( F l e i s c h e r e t a_l,
(Tzagoloff
dehydrogenase
essential for
substrate
mitochondria
W i t h b r a i n AChE t h e s e d i f f i c u l t i e s
are
compounded b y
lack of
precise
knowledge as
the
enzyme i n the
c e n t r a l nervous system.
to
the
action
I f i t i s assumed
of
66
that
c h o l i n e r g i c mechanisms i n t h e
neuromuscular
can
j u n c t i o n are
level
c e n t r a l nervous system
and
e s s e n t i a l l y s i m i l a r , some e s t i m a t e
be
made o f
the
a t w h i c h ACh
tained
f o r the
transmission
of
nerve
(1969) h a v e c a l c u l a t e d t h a t
the
i n t e r c o s t a l muscle c o n t a i n s
10
h y d r o l y s i s must be
impulses.
postsynaptic
main-
Namba and
Grob
membrane f r o m
rat
g
m o l e c u l e s o f AChE, and
that
one
_g
synapse h y d r o l y s e s
5 x 10
7
They e s t i m a t e
10
synaptic
that
space per
which could
be
m o l e c u l e s o f ACh
m o l e c u l e s o f ACh
are
hydrolysed
within
synaptic
membrane were known, i t w o u l d be
ACh
within
latency
period
time of
arrival
synaptic
ACh
of
synaptic
is generally
the
longer,
i n the
at
C a l c u l a t i o n s b a s e d upon t h e
synaptic
was
in
t h a t ACh
the
from t h i s
synaptic
s e c o n d s as
hydrolysis,
e x c e s s o f AChE.
but
the
rate
space
one
that
Clearly this
i t does i n d i c a t e t h a t
the
of
10
post
molecules
that
the
the
time o f
0.3
the
the
t o make some
7
post-
t o 0.5
milli-
action is
50 m i l l i s e c o n d s
for
the
synapse.
f r e e d i f f u s i o n of
ACh
become n e g l i g i b l e a t t h e
millisecond of
release,
and
posti t
some b a r r i e r t o f r e e d i f f u s i o n e x i s t s
f o r the
a r r i v e s a t an
to
Renshaw c e l l
of
to cross
measured from
transmitter
of
the
(1957) s t a t e s
order
( E c c l e s , 1957).
extreme v a l u e s
one
of
l e v e l s should
membrane w i t h i n
concluded
impulse
order
by
to hydrolyse
as
the
substrate
action at
possible
Eccles
in'the
duration
mediated transmission
indicated
transmitter
transmission
of a presynaptic
However,
considerably
of
time a v a i l a b l e .
response
seconds.
duration
amount o f AChE r e q u i r e d
the
into
t i m e t a k e n f o r ACh
space,
of
the
I f the
of
milliseconds
synaptic
the
and
0.02
millisecond.
released
action potential, a quantity
e s t i m a t e d a v a i l a b l e AChE.
estimate of
per
U s i n g 0.5
to
50
time a v a i l a b l e f o r
e s t i m a t e d 2 5 t o 2 500
milliACh
fold
i s a rough c a l c u l a t i o n a t
postsynaptic
AChE may
never
functional
best,
67
be
saturated with
concentrations
conclusions
(Srere,
serve
a t o r b e l o w the
V e g o t s k y and
to u n d e r l i n e
i n the
There
evidence
system.
the
Frieden,
importance
example,
Glow and
at
this
1963).
If there
central
nervous
stabilizing
level
A
Similar
data
in vivo.
w h i c h c o u l d be
c e n t r a l nervous
(1966) have shown i n
do
not
increase
b e e n r e d u c e d b e l o w 40
o f AChE i n h i b i t i o n
advantages
and
stabilization
of
too
compelling
t o be
data
accommodation o f
a t hand,
reaction rate
warm a c c l i m a t i o n s t a t e s i t may
same r a t e s o f ACh
In
conduction
(Wilson
and
as
be
point
trout
in
values,
thermally
f o r the
probably
induced
Km
changes
t r o u t AChEs,
seem
simply c o i n c i d e n t a l .
i t i s proposed that while
i s necessary
n o t be
situation,
Cohen,
i n the r e l a t i o n s h i p s
i n both
necessary
hydrolysis at different
this
may
inherent
reaction rates
dismissed
to
temperature f l u c t u a t i o n s .
minimum Km
concentrations,
value
interpreted
i s a f u n c t i o n a l e x c e s s o f AChE i n t h e
adaptive
systems
estimates
s h a r p d r o p i n neve
p h y s i o l o g i c a l ACh
temperatures.
enzyme
These
s y s t e m t h e r e w o u l d seem l i t t l e
From the
ACh
enzyme-substrate
levels
reaction rates during
the
minimum Km
of
AChE a c t i v i t y has
between h a b i t a t temperature,
the
enzyme.
1958).
Rose
r a t b r a i n t h a t ACh
of normal v a l u e s .
a l s o occurs
However,
o f the
o f a f u n c t i o n a l e x c e s s o f AChE i n the
of
percent
Km
i s exposed to
r e g u l a t i o n o f enzyme a c t i v i t i e s
significantly until
50
rather,
i s some e x p e r i m e n t a l
For
bioassays
but
have been a r r i v e d a t f o r m i t o c h o n d r i a l
1968;
affinities
as
substrate
to
i n vivo
cold
maintain
acclimation
selection for a
d e t e r m i n e d by
the
thermal
particular
substrate
and
68
concentrations,
physiological
substrate
c.
f o r i t i s o n l y when Km
substrate
concentration
levels
can
The
the
adaptation
inhabiting
different
that small
effectively
Evolutionary adaptation
(i) Adjustment of
to
a t or
regulate
t h e r m a l accommodation
enzyme
s u g g e s t e d f o r the
Km
values
may
thermal environments appears
by
the
species
based
will
the
species.
s e l e c t i o n of a
determined p r i m a r i l y by
in
t o be
r e l a t i o n s h i p that
experienced
t r o u t enzymes,
be
activity.
range
o f AChE f u n c t i o n t o t e m p e r a t u r e
range n o r m a l l y
below
fluctuations in
t h e r m a l accommodation f o r r e a c t i o n r a t e o v e r
temperature
of
are
temperature
upon s e l e c t i o n f o r a Km-temperature
allow
values
As
particular
range
p h y s i o l o g i c a l ACh
concentration.
If
thermally
shifts
the
induced
i n the
readily
conformational
Km-temperature
f r o m the
gradual
electric
diverged
data
similarities
between the
are
during
an
this hypothesis
the
similar
enzyme m o l e c u l e ,
s p e c i a t i o n can
be
conformation
advantage a t
this
p o i n t to
t h e v i e w t h a t the
present
trout
i n f a c t h o m o l o g o u s enzymes w h i c h h a v e
best
I n the
t h a t can
be
AChEs w h i c h a r e
o f a common o r i g i n .
e e l AChEs h a v e m o l e c u l a r
probably
upon
o f amino a c i d r e p l a c e -
f r o m a common a n c e s t r a l gene.
however,
electric
curve
to support
e e l AChEs a r e
information,
with
c h a n g e s i n the
accumulation
C l e a r l y i t w o u l d be
amino a c i d s e q u e n c e
and
r e l a t i o n s h i p i s dependent
i n t e r p r e t e d i n terms o f c h a n g e s i n
resulting
ments.
Km-temperature
i n net
absence of
done i s t o
at
Both
list
compatible
trout
and
w e i g h t s o f a b o u t 260,000,
and
c h a r g e and
1.
least
such
the
c o n f i g u r a t i o n as
judged
from e l e c t r o p h o r e s i s
conclusive,
b o t h the
2.
The
t h i s data
trout
and
Arrhenius
and
plots
temperatures.
are
the
the
Evolution
low
While
account
the
be
the
two
that
the
two
enzymes w o u l d be
duplication,
the
eel
properties
of
followed
1963;
has
trout
salmonids a t
A and
B
higher
enzyme
that
hydrolysis
a single
CNS.
One
1968;
presence
explanation
gene d u p l i c a t i o n ,
relationships
1964;
Watts,
Cohen and
1968;
duplicated
that
the
to
Gene
fates
(Smithies et a l ,
Watts,
1969).
l o c i are
known;
(Holmes and
Markert,
1969;
( C o r y and
1962;
1967;
genetic
1968), e n o l a s e
of
o r i g i n a l divergence
Milstein,
W a t t s and
is
and
for
independent e v o l u t i o n a r y
i n the
of
replacements.
by
l e a s t four
Markert,
the
t h e i r thermal ranges.
the
will
enzyme,
extend
l a c t a t e dehydrogenases
M a s s a r o and
same f o r m ,
properties
t h r o u g h amino a c i d
been i m p l i c a t e d
Rutter,
Augustinsson,
curved
AChEs.
o f many s t r u c t u r a l l y h o m o l o g o u s p r o t e i n s
Ingram,
structures.
b a s e d upon a d v a n t a g e s a c c r u i n g
could
duplicates,
The
proposed to e x p l a i n
d i f f e r e n t Km-temperature
i n d i v i d u a l s who
that
inhibitor
mechanisms o f
t r o u t enzymes a r o s e b y
of
proposition
activation at
t r o u t and
i n the
they subsequently diverged
Selection
means
r a i n b o w t r o u t b r a i n AChE complex
i n the
AChE v a r i a n t s
two
no
a c c u m u l a t i o n o f amino a c i d s u b s t i t u t i o n s
f o r changes
the
and
argued from these
f u r t h e r mechanisms must be
the
the
relationships.
e n e r g i e s of
s i t e s and
of
with
W h i l e by
enzymes h a v e e s s e n t i a l l y the
p r o b a b l y s i m i l a r i n the
(ii)
gels.
s i m i l a r substrate
I t can
active
is consistent
pH-activity
for
approaching very
acrylamide
e e l enzymes h a v e s i m i l a r s u b u n i t
AChEs d i s p l a y
specificities
both
on
Wold,
1966),
In
and
supernatant malate
dehydrogenase
( B a i l e y e t a l , 1969),
and
r e c e n t l y m u l t i p l e forms o f s e v e r a l enzymes i n a d d i t i o n
have been found
Somero a n d
i n the
Hochachka,
1970).
In f a c t ,
entire
genome has
about
rainbow t r o u t
1968;
i t appears
Somero,
likely
1969;
H o c h a c h k a and
fish
per n u c l e u s and
as
i n the
Atkin,
Hinegardner,
is
same o r d e r
c o n s i d e r a b l e s u p p o r t f o r the view t h a t
arose
following
An
may
of
Ohno e t a l , 1968;
the
alternative hypothesis
gene d u p l i c a t i o n ,
i s that
following
two
type
have
l e d t o the
t h e one
presence
individual.
genotype
populations at a
o f the
two
have a r i s e n
species
There
hybridization
possessed
that
from both
Bouck a n d
Ball
lead
later
time
regimes.
might
to favour
salmonids
may
diploid
f o r a number o f
enzymes.
the view
incorporation
that speckled
27
this
Markert
p a r e n t s w i t h i n the h y b r i d .
at least
of
i n the p o p u l a t i o n ,
to support
t o the
(1968) f o u n d
F, h y b r i d s p o s s e s s e d
AChEs
absence
thermal
Massaro and
different alleles
among t r o u t c a n
there
t r o u t AChEs
the t e t r a p l o i d
i s experimental evidence
enzyme v a r i a n t s
trout
trout
from h y b r i d s formed between a n c e s t r a l
that
example,
suggested
as
and
Thus
i n the
I f c o n d i t i o n s were s u c h a s
i t m i g h t have b e e n e s t a b l i s h e d
in fact
twice
d i v e r g e n t enzymes w i t h i n
e v e n t u a l l y becoming the dominant form.
(1968) have
possess
the r e p r o d u c t i v e i s o l a t i o n
p o p u l a t i o n s t h a t were e x p o s e d t o d i f f e r e n t
I n t e r b r e e d i n g b e t w e e n the
Lewis,
gene.
the
a single molecular
two
1968;
the
(Ohno
1969).
d u p l i c a t i o n o f an a n c e s t r a l
have d i v e r g e d from
two
salmonids
approximately
many chromosomes as o t h e r f i s h
1966;
Somero,
t h a t d u p l i c a t i o n of
o c c u r r e d i n these
t w i c e as much DNA
(Hochachka and
t o AChE
that
of
For
trout-brown
electrophoretically
71
distinct
l a c t a t e dehydrogenases, w h i l s t o n l y
detected
i n each o f the p a r e n t a l
described
lake
a s i m i l a r s i t u a t i o n with
trout hybrid,
dehydrogenase
generations
An
is
being
(Ihssen,
a n d was a b l e
profile
lake
personal
species,
and s p l a k e
AChE s y s t e m
The
t o show t h a t
i n trout
t r o u t and
hybrids
lake
The h y b r i d ,
l y i n g b e t w e e n t h e two
between d i f f e r e n t
trout bybrids.
generations
of
I t was c o n s i d e r e d
of p a r t i c u l a r
t o the e v o l u t i o n o f the rainbow
f o r acrylamide
gel disc
o f b r a i n homogenates f r o m
groups o f
t r o u t and s p l a k e
After acclimation
P
hybrids
Following
t r o u t each give
acclimated
trout
fish.
speckled
to 4 ° ,
9°
1 8 , a n d c a n be s u m m a r i s e d as
to 4 ° c speckled
and l a k e
t r o u t each
h a s one m a j o r AChE b a n d a n d
acclimation
t o 9°C, s p e c k l e d
two AChE b a n d s , w h i l e
b a n d s a n d two m i n o r b a n d s .
been
speckled,
t o s t u d y t h e i n h e r i t a n c e o f AChEs i n t h e s e
two m i n o r b a n d s .
and
subsequent
and a range o f t h e r m a l t o l e r a n c e s have
r e s u l t s obtained
lake
Speckled
rolerance
show one AChE b a n d , w h e r e a s s p l a k e
trout
through
tolerance
upper thermal t o l e r a n c e s .
2 0 ° C a r e shown i n F i g u r e
lake
trout-
the l a c t a t e
was m a i n t a i n e d
communication).
repsect
electrophoresis
follows:
the speckled
conducted by Ihssen a t the U n i v e r s i t y o f Toronto
i n t e r e s t with
and
splake,
i n v e s t i g a t i o n o f temperature
i n crosses
trout,
Goldberg has
1966; G o l d b e r g e t a l , 1 9 6 7 ) .
has a temperature
parental
found
i n splake
(Goldberg,
t r o u t have d i f f e r e n t
splake,
stocks.
15 c o u l d be
splake
h a s two m a j o r
A f t e r 14 days a t 2 0 ° C ,
show a s i n g l e AChE band, w h i l e
one m i n o r b a n d o f AChE a c t i v i t y .
splake
and
speckled
h a s one m a j o r b a n d
Unfortunately,
20°C
lake
72
Figure
18.
Resolution
Trout,
o f B r a i n AChEs f r o m
Lake T r o u t
Gel Disc
and Splake by A c r y l a m i d e
Electrophoresis.
b a n d s were
Specific
AChE
i d e n t i f i e d by i n h i b i t i o n
with
284C51 a n d e s e r i n e .
only
Speckled
The d i a g r a m
shows
t h e number o f b a n d s r e s o l v e d a n d
does n o t i n d i c a t e t h e r e l a t i v e
migration
r a t e s o f the f r a c t i o n s , a p r o p e r t y
can
which
o n l y be a c c u r a t e l y d e t e r m i n e d b y
running
mixtures within
electrophoretic
technique
conditions
are given
Acclimation
4°C
fish
and
20°C f i s h
t h e one g e l . The
and s t a i n i n g
i n t h e methods s e c t i o n .
periods
f o r 6 weeks,
were a s f o l l o w s :
9°C f i s h
f o r 2 weeks.
f o r 6 weeks,
4 C acclimated trout
speckled
lake
splake
speckled
lake
splake
9°C acclimated trout
20°C
acclimated trout
speckled
splake
73
trout brains
were n o t
obtained.
were a v a i l a b l e i t was
mobilities
of
the
homogenates, o r
it
i s not
present
types,
the
the
formation
which are
t o an
hybrid.
a simple
i n the
Further,
the
number o f
the
number o f AChE
summation o f
the
that
interbreeding
types
or
between t r o u t
isolated
from
species
i n nature can
presence of s i m i l a r t h e r m a l l y
inducible
suggest that
the
original
i n c o r p o r a t i o n of m u l t i p l e
AChE
species
occurred
prior
divergence
of
these three
(iii)
While
may
underly
species,
the
switching
rates
during
the
evolutionary
composition of
thermal
one
to
types
mechanism must be
during
i t i s not
the
t r o u t b r a i n AChE
acclimation
gene d u p l i c a t i o n and
presence of m u l t i p l e
development o f
enzyme p r o f i l e
present
of
s u c h e v e n t s as
the
leads
trout.
Regulation
complex
to the
trout
the
i n rainbow,
one
lake
in
lead
AChE s y s t e m s
into
and
polypeptide
i t i s apparent
number o f AChE enzymes p r e s e n t
speckled
Thus,
parental
mechanism,
However,
brain
enzymes.
of h y b r i d molecules containing
AChE.
fish
the e l e c t r o p h o r e t i c
running mixtures of
i n the
normally reproductively
increase
t o compare
thermal switching
from each p a r e n t a l
r e s u l t s obtained
a small
characterize
increase
involves
breakdown o f
chains
only
d i f f e r e n t AChEs b y
known i f t h i s
the
possible
to k i n e t i c a l l y
i n splake
possibly
not
As
forms o f AChE w i t h i n
some f o r m o f
postulated
thermally
to explain
thermal a c c l i m a t i o n
of
p o s s i b l y even m o d i f i c a t i o n
enzyme d e g r e d a t i o n ,
of
the
gene p r o d u c t s
one
controlled
changes
trout.
known i f t h e s e c h a n g e s r e s u l t
o f enzyme s y n t h e s i s ,
hybridization
from
in
At
altered
or both,
or
following
74
synthesis,
t h u s any
d i s c u s s i o n of
AChE complex must be
value,
to
at
this
speculative.
point-
synthesis
Berlin,
e f f e c t s of
Jankawsky,
1969;
limiting
for future
have been s t u d i e d
(Mews, 19 57;
to
of
proteins
during
specific
Das
and
and
have been i d e n t i f i e d
relating
the
trout
rates
investigation.
protein
poikilotherms
Prosser,
probable
1967;
Dean
thermally
(Haschemeyer,
of
of
c o n t r o l mechanisms i f o n l y
t e m p e r a t u r e upon t o t a l
1969)
information
the
i t w o u l d seem
i n a v a r i e t y of
1960;
Haschemeyer,
steps
E v e n so,
to o u t l i n e p o s s i b l e
i n d i c a t e important areas
A l t h o u g h the
r e g u l a t i o n of
synthesis
rate
1969),
and
thermal a c c l i m a t i o n
and
degradation
is
completely
lacking.
If
the
acclimation
of
temperatures
protein synthesis,
properties
the
amounts o f e a c h AChE enzyme p r e s e n t
of
enzyme s y n t h e s i s
a l s o be
to
the
a t the
trout.
These
structure
and
possibilities
postulate
operator
level of
through
the
t r o u t system,
genes may
f r o m the
i n the
livers
have
thereby
thermally
Regulation
at
production.
With
communication)
profiles
o f warm and
of
reflect
function
thermal a c c l i m a t i o n .
include
the
amino a c y l t r a n s f e r a s e s
changes i n
modulation of
a rate
rainbow
ribosomal
Other
s u c h enzymes as
w h i c h a p p e a r t o be
has
ribosomal
cold acclimated
d i f f e r e n c e s may
during
accompanied
i n d u c e d changes
(person
melting
the
regulating
transcription.
Somero
regulation
t h a t changes i n
s u b s e q u e n t t o m e s s e n g e r RNA
found c l e a r d i f f e r e n c e s
preparations
can
d e t e r m i n e d by
s t r u c t u r a l genes,
achieved
numerous s t e p s
reference
one
r e g u l a t o r and
divergence of
could
i n t r o u t are
at particular
the
limiting
step
75
in
protein
fish
synthesis
following
t o lower temperatures
Alternately,
preformed p r o t e i n
t h e t r a n s f e r o f warm
(Haschemeyer,
1969).
the a c t i v a t i o n and i n a c t i v a t i o n o f
through m o d i f i c a t i o n
a d d i t i o n a n d r e m o v a l o f amino a c i d s ,
composition could
acclimated
o f amino a c i d s i d e
o r changes i n s u b u n i t
be i m p o r t a n t methods o f r e g u l a t i o n .
Thermal s w i t c h i n g
o f t h e 'warm' a n d ' c o l d ' AChEs i n
enzyme m e d i a t e d mechanisms m i g h t be a c h i e v e d d i r e c t l y
Km-temperature
r e l a t i o n s h i p s s i m i l a r t o those
i n hormone b a l a n c e .
The i n f l u e n c e
and a c t i v i t y
1964;
induced
changes
o f hormones i n c o n t r o l l i n g
o f enzymes i s w e l l
( M e t z e n b e r g e_t a l , 1961; McKearns,
through
observed with the
AChEs, o r i n d i r e c t l y b y s u c h f a c t o r s a s t h e r m a l l y
both synthesis
groups,
established
1963; V a r n e r a n d Ramchandra,
Kim e t a l , 1966; O k i e t a l , 1966; Tomkins e t a l , 1 9 6 9 ) .
It
complexes
i s hoped t h a t
i n poikilotherms
system f o r s t u d i e s
vertebrates.
these
will
thermally
controled
provide a valuable
o f the r e g u l a t i o n
enzyme
experimental
o f gene e x p r e s s i o n
in
76
SUMMARY
The
system
effects
of temperature
t h e r m a l a c c l i m a t i o n and
a d a p t a t i o n to temperature
At
o f ACh
level
nervous
interpreting
evolutionary
o f enzyme
function.
concentrations,
the
rate
h y d r o l y s i s b y AChE c a n r e m a i n r e l a t i v e l y u n a f f e c t e d
throughout a temperature
t h a t e x p e r i e n c e d by
P l o t s o f Km
rainbow
versus temperature
r
difference
temperature
that
from
curves with
close
sucrose gradient
spectra
probably r e s u l t
enzyme c o n f o r m a t i o n , and
f o r AChE enzymes
temperatures
Studies u t i l i z i n g
ultraviolet
range c o r r e s p o n d i n g
e e l y i e l d U shaped
values occurrihg at
temperatures.
by
the a n i m a l i n n a t u r e .
t r o u t and e l e c t r i c
minimum Km
with
a t the
p r o b a b l e p h y s i o l o g i c a l ACh
assay temperature
and
the
o f f i s h were i n v e s t i g a t e d w i t h the o b j e c t o f
t h e r m a l accommodation,
to
upon AChE f r o m
t o the
habitat
centrifugation
s u g g e s t t h a t changes i n
from s m a l l a l t e r a t i o n s
sharp breaks
i n the
Km
in
Km-temperature
curve are not a s s o c i a t e d w i t h changes i n m o l e c u l a r a g g r e g a t i o n
or
the d i s s o c i a t i o n o f
Energies
subunits.
of a c t i v a t i o n
f o r the t r o u t and e l e c t r i c e e l
enzymes d e c r e a s e w i t h i n c r e a s i n g
relationship with habitat
It
substrate
the o b s e r v e d t h e r m a l
range
clear
accommodation
plots
temperature.
o f l o g Vdptversus
and e e l AChEs c o n t i n u e t o i n c r e a s e
temperature
show no
i s a c h i e v e d t h r o u g h c h a n g e s i n enzyme
a f f i n i t y with
Arrhenius
trout
rate
and
temperature.
i s concluded that
o f AChE r e a c t i o n
temperature
e x p e r i e n c e d by
1/T
f o r the
throughout
rainbow
the
the a n i m a l s , i n d i c a t i n g
that
77
thermal
limits
denaturation i s probably not a f a c t o r
f o r t h e r m a l accommodation.
reached
It
f o r AChE f r o m
i s proposed
important
that
the A n t a r c t i c
i n s e t t i n g both
stabilization
Thermal
trout,
and
and
upper
the r e l a t i v e
displaying
different
second
is
no
better
lower
suited
substrate
associated with
enzyme a c t i v i t y .
of these
of the rainbow
variants
When t h e
where one
activity
form
for reaction
i s lost,
a
relationship
functions.
trout,
b o t h AChE
Thus a t p r o b a b l e
c a t a l y s e d by
proceed a t a slower rate
'warm' enzyme a t 17°C,
variants
physiological
the
than the
'cold'
reaction
unless other
factors
the c o l d a c c l i m a t i o n p r o c e s s a c t to i n c r e a s e
With
this
pH a n d membrane
considered.
I t was
and
AChE
by
v a l u e s a l t h o u g h t h e s e minima o c c u r a t
environment,
environmnet
i s achieved
relationships.
c o n c e n t r a t i o n s the r e a c t i o n
the
rainbow
f o r w h i c h t h e Km-temperature
temperatures.
enzyme a t 2°C w i l l
i n the
t o a range
between
regulation.
lake trout,
is altered
for control
different
c a t a l y s e d by
interaction
p r o p o r t i o n s o f two
be
f o r thermal
l o n g e r t h e r m a l l y accommodate
h a v e s i m i l a r minimum Km
markedly
limits
through an
Km-temperature
i s produced
In t h e c a s e
lipids
r e l a t i o n s h i p may
o r where r e g u l a t i o n o f c a t a l y t i c
form
c o n c l u s i o n was
t h e m a i n t e n a n c e o f enzyme
temperature
the enzyme c a n
rate,
and
upper
Trematomus b o r c h g r e v i n k i .
a c c l i m a t i o n o f AChE a c t i v i t y
regulating
of
fish
p r o b a b l y i n s p e c k l e d and
environmental
similar
t h e Km-temperature
a c c o m m o d a t i o n o f AChE a c t i v i t y
rate
A
in setting
found
pH may
be
i n mind,
lipids
the e f f e c t s
of importance,
no
ionic
upon AChE a c t i v i t y
t h a t w h i l e changes
i n t h e enzyme e x t r a c t h a d
of
i n both
were
ionic
the presence
significant effect
o f membrane
upon
AChE a c t i v i t y AChE f r o m c o l d
that
the
rate
Determinations
(2°C) a n d warm
compensation
total
of specific
I t i s concluded
rate
t h e c o l d a n d warm a c c l i m a t i o n
n e c e s s a r y t o m a i n t a i n t h e same r a t e s
acclimation
The
i s probably
states
be
b a s e d upon s e l e c t i o n f o r a _Km-temperature
t h e Km-temperature
in
terms o f c h a n g e s
curve
during
speciation
i n enzyme c o n f o r m a t i o n
a c c u m u l a t i o n o f amino a c i d s u b s t i t u t i o n s .
i s p r e s e n t e d which, w h i l e
the view t h a t
derived
Possible
rate
into
considered.
While
gene d u p l i c a t i o n
the t r o u t
there
i n this
involving hybridization
This
that
over the
Shifts
are interpreted
Physical
not conclusive,
the gradual
and k i n e t i c
i s consistent
e e l AChEs were
gene.
mechanisms b y w h i c h two AChE enzymes c o u l d
incorporated
hybrids,
relationship
following
rainbow t r o u t and e l e c t r i c
from-, common a n c e s t r a l
suggested.
t o temperature
range n o r m a l l y e n c o u n t e r e d by the s p e c i e s .
in
with
i t may n o t be
d i f f e r e n t thermal environments appears t o
a l l o w t h e r m a l accommodation o f r e a c t i o n
evidence
necessary
a d a p t a t i o n o f AChE f u n c t i o n
species
temperature
although
o f AChE h y d r o l y s i s a t
in
will
that
temperatures.
evolutionary
inhabiting
indicated
i s not achieved through a l t e r a t i o n s i n
amount o f AChE p r e s e n t .
different
f o r brain
(17°C) a c c l i m a t e d t r o u t
t h e r m a l accommodation o f r e a c t i o n
i n both
activity
c e n t r a l n e r v o u s s y s t e m were
i s considerable
process,
an a l t e r n a t i v e
between f i s h
that
which are normally r e p r o d u c t i v e l y
trout
hypothesis
also
interspecies
c r o s s e s between t r o u t
i s o l a t e d could
also
implicating
p o p u l a t i o n s was
t h e o r y was e x a m i n e d w i t h
a n d i t was o b s e r v e d
evidence
be
species
r e s u l t i n an
79
increased
the
number o f AChE enzymes p r e s e n t
p r e s e n c e o f s i m i l a r AChE c o m p l e x e s
speckled
and lake
trout indicated that
o f m u l t i p l e AChEs i n t o one s p e c i e s
the
evolutionary
It
divergence
can
Further,
trout,
the o r i g i n a l
occurred
incorporation
prior to
trout.
study
thermal a c c l i m a t i o n and e v o l u t i o n a r y
as
i n rainbow
probably
o f these
i s concluded from t h i s
i n the h y b r i d .
that
thermal
adaptation
d i s p l a y e d b y many p h y s i o l o g i c a l s y s t e m s
accommodation,
t o temperature
i n poikilotherms
be o b s e r v e d a n d i n t e r p r e t e d a t t h e l e v e l
o f enzyme
function.
80
ABBREVIATIONS
ACh
-
acetylcholine
AChE
-
acetylcholinesterase
ChAc
-
choline
Ea
-
energy o f a c t i v a t i o n
°K
-
degrees
Km
-
Michaelis
&0.D.
-
change i n o p t i c a l
T
-
absolute
tris
-
tris(hydroxymethyl)aminomethane
Vmax
-
maximum v e l o c i t y
Vopt
-
optimum v e l o c i t y
284C51
-
d i m e t h o b r o m i d e o f 1:5 -
acetyltransferase
absolute
constant
density
temperature
aminophenyl)-pentan-3-one
di(p-N-allyl-N-methyl-
81LITERATURE CITED
A l e x a n d r o v e , V. Y.
1969.
Conformational f l e x i b i l i t y of
p r o t e i n s and t h e i r r e s i s t a n c e t o p r o t e i n a s e s
and
temperature c o n d i t i o n s of l i f e .
C u r r e n t s i n Modern
B i o l o g y 3:
9-19.
Allen,
R. C ,
R. A. Popp, a n d D. J . Moore.- 1965.
Separation
and r e l a t i v e q u a n t i t a t i o n o f mouse p l a s m a e s t e r a s e s w i t h
disc electrophoresis.
J . H i s t o c h e m . C y t o c h e m . 13:
249-254.
A p r i s o n , M. H., R. T a k a h a s h i , and T. L. F o l k e r t h .
1964.
B i o c h e m i s t r y o f the a v i a n c e n t r a l n e r v o u s s y s t e m - 1.
The 5 - h y d r o x y - t r y p t o p h a n d e c a r b o x y l a s e ^ m o n o a m i n e
o x i d a s e and c h o l i n e - a c e t y l a s e - a c e t y l c h o l i n e s t e r a s e
s y s t e m s i n s e v e r a l d i s c r e t e a r e a s o f the p i g e o n b r a i n .
J . Neurochem. 11:
341-350.
A r m e t t , C. J . and J V M .
Ritchie.
1960.
The a c t i o n o f
a c e t y l c h o l i n e on c o n d u c t i o n i n mammalian n o n - m y e l i n a t e d
f i b r e s and i t s p r e v e n t i o n b y an a n t i c h o l i n e s t e r a s e .
J . P h y s i o l . 152: 141-158.
A t k i n s o n , D. E .
1966.
Rev. B i o c h e m . 35:
Regulation
85-124.
o f enzyme a c t i v i t y .
Ann.
A u g u s t i n s s o n , K-B.
1957.
A s s a y methods f o r c h o l i n e s t a s e s ,
p. 1-63..In "Methods o f B i o c h e m i c a l A n a l y s i s " V o l . 5.
(G. G l i c k , e d . ) .
Interscience Publ.
N.Y.
A u g u s t i n s s o n , K-B.
1968.
The e v o l u t i o n o f e s t e r a s e s i n
v e r t e b r a t e s , p. 299-311.
I n "Homologous Enzymes a n d
Biochemical Evolution".
(N. Van T h o a i and J . Roche,
Gordon and B r e a c h , L o n d o n .
ed.)
A u s t i n , L. and W. K. B e r r y .
1953.
Two s e l e c t i v e i n h i b i t o r s o f
cholinesterase.
B i o c h e m . J . 54: 695-700.
B a i l e y , G. S., G. T. C o c k s and A. C. W i l s o n .
1969.
Gene
d u p l i c a t i o n i n f i s h e s : Malate dehydrogenases of salmon
and t r o u t .
B i o c h e m . B i o p h y s . Res. Commun. 34: 605-612.
Baslow, M. H.
1967.
T e m p e r a t u r e a d a p t a t i o n i n the c e n t r a l
n e r v o u s s y s t e m o f f i s h , p. 205-22 6.
I n " M o l e c u l a r Mechanisms
o f Temperature A d a p t a t i o n " .
(C. L. P r o s s e r , ed.) Amer.
A s s . A d v a n c e . S c i . P u b l . No. 84.
Horn-Shafer,
Baltimore.
B a s l o w , M. H. and R. F. N i g r e l l i .
1964.
The e f f e c t o f t h e r m a l
a c c l i m a t i o n on b r a i n c h o l i n e s t e r a s e a c t i v i t y o f the
k i l l i f i s h , F u n d u l u s h e t e r o c l i t u s . Z o o l o g i c a 149: 41-51.
82
B a t t l e , H. I . 192 6.
E f f e c t s o f extreme temperatures on muscle
and nerve t i s s u e i n marine f i s h e s .
Roy. S o c . Can. T r a n s .
S e r . 3. 2 0 : 127-143.
B a t t l e , H. I . 1929. A n o t e o n l e t h a l
with skate r e f l e x e s .
Can. B i o l .
4: 497-500.
temperature
Fisheries-,
i n connection
Contrib.
B e h r i s c h , H. W.
1969. T e m p e r a t u r e a n d t h e r e g u l a t i o n o f
enzyme a c t i v i t y i n p o i k i l o t h e r m s . F r u c t o s e d i p h o s p h a t a s e
from m i g r a t i n g salmon.
B i o c h e m . J . 115: 687-696.
B e h r i s c h , H. W. a n d P. W. H o c h a c h k a .
1969. T e m p e r a t u r e a n d
t h e r e g u l a t i o n o f enzyme a c t i v i t y i n p o i k i l o t h e r m s .
Properties o f lungfish fructose diphosphatase.
Biochem.
J . 112: 601-607.
B e n n e t t , E . L., M. C. Diamond, D. K r e c h , a n d M. R. R o s e n z w e i g .
1964.
Chemical and anatomical p l a s t i c i t y o f b r a i n .
S c i e n c e 146: 610-619.
B e r n s o h n , J . , K. D. B a r r o n , a n d M. T. H e n d r i e k .
1963. Some
p r o p e r t i e s o f isoenzymes o f b r a i n a c e t y l c h o l i n e s t e r a s e .
B i o c h e m . P h a r m a c o l . 12: 761-764.
Bouck,
G. R. a n d R. C. B a l l .
1968. C o m p a r a t i v e e l e c t r o p h o r e t i c
p a t t e r n s o f l a c t a t e dehydrogenase i n t h r e e s p e c i e s o f
trout.
J . F i s h . R e s . B o a r d C a n . 2 5 : 1323-1331.
Bowen, W. J . a n d T. D. K e r w i n .
1956. The k i n e t i c s o f m y o k i n a s e .
II.
Studies o f heat d e n a t u r a t i o n , the e f f e c t s o f s a l t s
and t h e s t a t e o f e q u i l i b r i u m .
A r c h . Biochem. B i o p h y s .
64: 278-284.
B r e c h t , F . C.
1908. Some o b s e r v a t i o n s o n t h e n a t u r e o f h e a t
p a r a l y s i s i n nervous t i s s u e .
Amer. J . P h y s i o l . 2 2 :
456-476.
Brett,
J . R.
1956. Some p r i n c i p l e s i n t h e t h e r m a l
of f i s h .
Q u a r t . Rev. B i o l . 3 1 : 75-87.
requirements
Brzin,
M.
1966. The l o c a l i z a t i o n o f a c e t y l c h o l i n e s t e r a s e i n
a x o n a l membranes o f f r o g n e r v e f i b e r s .
Nat. Acad. S c i . ,
P r o c . 56: 1560-1563.
Bull,
D. L . a n d D. A. L i n d q u i s t .
1968. C h o l i n e s t e r a s e i n b o l l
w e e v i l s , Anthonomus g r a n d i s Boheman - 1. D i s t r i b u t i o n
a n d some p r o p e r t i e s o f t h e c r u d e enzyme.
Comp. B i o c h e m .
P h y s i o l . 2 5 : 639-649.
B u r g e n , A. S. V. a n d L . M. Chipman.
1951. C h b l i
t e r a s e and
s u c c i n i c dehydrogenase i n the c e n t r a l nervous system o f
t h e d o g . J . P h y s i o l . 114: 296-305.
n e s
83
C e l e s i a , G. G. a n d H. H. J a s p e r .
1966.
Acetylcholine released
from the c e r e b r a l c o r t e x i n r e l a t i o n t o s t a t e o f a c t i v a t i o n .
N e u r o l o g y 16: 1053-1063.
Chadwick, L . E .
1957.
T e m p e r a t u r e dependence o f c h o l i n e s t e r a s e
a c t i v i t y , p. 45-59.
I n " I n f l u e n c e o f T e m p e r a t u r e on
B i o l o g i c a l Systems".
( F . H. J o h n s o n , ed.) W a v e r l y
Press, Inc., Baltimore.
Changeux, J - P .
1966.
Responses
T o r p e d o marmorata t o s a l t s
P h a r m a c o l . 2: 369-392.
o f a c e t y l c h o l i n e s t e r a s e from
and c u r a r i z i n g d r u g s .
Mol.
Changeux, J-P., W. L e u z i n g e r , a n d M. H u c h e t .
1968.
Specific
b i n d i n g o f a c e t y l c h o l i n e t o a c e t y l c h o l i n e s t e r a s e i n the
presence of e s e r i n e .
F.E.B.S. ' L e t t e r s 2: 77-80.
Changeux, J-P., T. P o d l e s k i , a n d J . C. M e u n i e r .
1969.
On some
s t r u c t u r a l a n a l o g i e s between a c e t y l c h o l i n e s t e r a s e a n d
the m a c r o m o l e c u l a r r e c e p t o r o f a c e t y l c h o l i n e , p. 22 5244.
i n "Membrane P r o t e i n s " .
New Y o r k H e a r t A s s o c i a t i o n
Symposia.
L i t t l e , Brown a n d Co.:, B o s t o n .
Cheeseman, D. F., W. C. Lee, a n d P. F. Z a g a l s k y .
1967.
Carote no p r o t e i n s i n i n v e r t e b r a t e s .
B i o l . Rev. 42:
160.
131-
Cohen,
S., a n d C. M i l s t e i n . 1967.
Structure
molecules.
N a t u r e 214: 449-452.
of antibody -
Cory,
R. P., a n d F. W o l d .
1966.
I s o l a t i o n and c h a r a c t e r i z a t i o n
o f e n o l a s e f r o m r a i n b o w t r o u t (Salmo g a i r d n e r i i g a i r d n e r i i )
Biochemistry
5: 3131-3137.
Cotman, C , M. L. B l a n k , A. M o e h l , a n d F. S n y d e r .
1969.
L i p i d c o m p o s i t i o n o f s y n a p t i c p l a s m a membranes
i s o l a t e d from r a t b r a i n by z o n a l c e n t r i f u g a t i o n .
B i o c h e m i s t r y 8: 4606-4612.
Das,
A. B. a n d C. L. P r o s s e r .
1967.
B i o c h e m i c a l changes i n
t i s s u e s o f g o l d f i s h a c c l i m a t e d t o h i g h a n d low tempe r a t u r e s - 1.
Protein synthesis.
Comp. B i o c h e m .
P h y s i o l . 21: 449-467.
Davis,
B. J .
1964.
D i s c e l e c t r o p h o r e s i s - I I . Methods a n d
a p p l i c a t i o n t o human serum p r o t e i n s .
A n n a l s . . N. Y.
A c a d . S c i . 121: 404-427.
Dean, J . M., a n d J . D. B e r l i n .
1969.
A l t e r a t i o n s i n hepatocyte
f u n c t i o n o f t h e r m a l l y a c c l i m a t e d r a i n b o w t r o u t (Salmo
g a i r d n e r i i ) . Comp. B i o c h e m . P h y s i o l . 29: 307-312.
De
R o b e r t i s , E.
secretion.
1964.
H i s t o p h y s i o l o g y o f synapses and
Pergamon P r e s s ,
London.
neuro-.
84
D e t t b a r n , W. D.
1960a.
E f f e c t o f c u r a r e on c o n d u c t i o n i n
myelinated, i s o l a t e d nerve o f the f r o g .
N a t u r e 186:
891-892.
D e t t b a r n , W. D.
1960b.
New e v i d e n c e f o r t h e r o l e o f
acetylcholine i n conduction.
Biochim. Biophys.
41: 377-386.
Acta
Dure11, J . , J . T. G a r l a n d , a n d R. 0. F r i e d e l .
1969.
Acetylcholine action:
biochemical aspects.
S c i e n c e 165:
862-866.
E c c l e s , J . C.
1957. The P h y s i o l o g y o f N e r v e C e l l s .
Hopkins Press, B a l t i m o r e .
E c c l e s , J . C.
1964. The P h y s i o l o g y o f S y n a p s e s .
Verlag, B e r l i n .
John
Springer-
E c c l e s , J . C , P. F a t t , a n d K. K o k e t s u .
1954. C h o l i n e r g i c
a n d i n h i b i t o r y s y n a p s e s i n a pathway f r o m m o t o r - a x o n
c o l l a t e r a l s t o motorneurones.
J . P h y s i o l . 126: 524562.
E h r e n p r e i s , S.
1967. M o l e c u l a r a s p e c t s o f c h o l i n e r g i c
mechanisms, p. 1-78. I n "Drugs A f f e c t i n g t h e P e r i p h e r a l
Nervous System".
Med. R e s . S e r . Monogr. v o l . 1.
M a r c e l D e k k e r , I n c . , N. Y.
Fast,
P. G.
1967. E t h a n o l a m i n e p h o s p h o g l y c e r i d e s :
on t h e p r o p e r t i e s o f m y e l e n o i d l e c i t h i n w a t e r
S c i e n c e 155: 1680-1681.
effect
systems.
F e l d b e r g , W.
1945. P r e s e n t v i e w s o n t h e mode o f a c t i o n o f
a c e t y l c h o l i n e i n the c e n t r a l nervous system.
Physiol.
Rev. 2 5 : 596-642.
Feldberg, W., a n d M. V o g t .
1948. A c e t y l c h o l i n e s y n t h e s i s i n
d i f f e r e n t r e g i o n s o f the c e n t r a l nervous system. J .
P h y s i o l . 107: 372-381.
F i s c u s , W. G. a n d W. C. S c h n e i d e r .
phosphoryl-choline - c y t i d y l
P r o c . 24: 4 7 6 . ( A b s t r . )
1965. A c t i v a t i o n o f
transferase. Federation
F i s h e r , K. C.
1958. An a p p r o a c h t o t h e o r g a n a n d t h e c e l l u l a r
a d a p t a t i o n t o t e m p e r a t u r e i n f i s h a n d s m a l l mammals,
p. 3-49. I n " P h y s i o l o g i c a l A d a p t a t i o n " (C. L . P r o s s e r ,
ed.).
Amer. P h y s i o l . S o c . W a s h i n g t o n , D.C.
F l e i s c h e r , S., G. B r i e r l e y , H. Klouvasn, a n d D. B. S l a u t t e r b a c k .
1962.
Studies o f the e l e c t r o n t r a n s p o r t system.
XLVII.
The r o l e o f p h o s p h o l i p i d s i n e l e c t r o n t r a n s f e r .
J . B i o l . Chem. 237: 3264-3272.
85
Foss,
J . G.
1960. T e m p e r a t u r e d i f f e r e n c e
B i o p h y s . R e s . Commun. 2: 193-197.
Foss,
J . G.
1961. H y d r o p h o b i c b o n d i n g a n d c o n f o r m a t i o n a l
t r a n s i t i o n s i n lysosyme, r i b o n u c l e a s e and c h y m o t r y p s i n .
B i o c h i m . B i o p h y s . A c t a 4 7 : 569-579.
Fry,
spectra.
Biochim.
F . E . J . 1947. E f f e c t s o f e n v i r o n m e n t o n a n i m a l
Ontario F i s h e r i e s Lab. Publ.
55: 1-62.
activity.
Glow,
P. H. a n d S. R o s e .
operant e x t i n c t i o n .
165-172.
Glow,
P. H., S. Rose, a n d A. R i c h a r d s o n . . 1966. The e f f e c t o f
acute and c h r o n i c treatment w i t h d i i s o p r o p y l f l u o r o p h o s p h a t e o n c h o l i n e s t e r a s e a c t i v i t i e s o f some t i s s u e s
o f t h e r a t . A u s t . J . E x p . B i o l . Med. S c i . 44: 73-86.
G o l d b e r g , E . 1966.
hybridization
1091-1093.
1966. C h o l i n e s t e r a s e l e v e l s a n d
J . Comp. P h y s i o l . P s y c h o l . 61:
L a c t a t e dehydrogenase
i n v i v o and i n v i t r o .
i n trout:
S c i e n c e 151:
G o l d b e r g , E., J - P . C u e r r i e r , a n d J . C. Ward.
1967. L a c t a t e
d e h y d r o g e n a s e i s o z y m e s , v e r t e b r a e a n d c a e c a numbers i n
an
isolated, interbeeding population o f splake trout.
N a t u r a l i s t e Can. 94: 297-304.
G r a f i u s , M. A., S. L . F r i e s s , a n d D. B,. M i l l a r .
1968.
of the p o l y d i s p e r s i t y o f a c e t y l c h o l i n e s t e r a s e by
t r a n s p o r t methods i n t h e u l t r a c e n t r i f u g e .
Arch.
B i o c h e m . B i o p h y s . 126: 707-721.
Analysis
Haschemeyer, A. E . V. 1969. S t u d i e s o n t h e c o n t r o l o f p r o t e i n
s y n t h e s i s i n low t e m p e r a t u r e a c c l i m a t i o n .
Comp. B i o c h e m .
P h y s i o l . 2 8 : 535-552.
H a s s o n - V o l o c h , A.
1968. C u r a r e a n d a c e t y l c h o l i n e
substance.
N a t u r e 218: 330-333.
receptor
Hebb, C. 1963. F o r m a t i o n , s t o r a g e a n d l i b e r a t i o n o f
a c e t y l c h o l i n e , p. 55-88.
I n "Handbuch d e r E x p e r i m e n t e l l e n
Pharmakologie".
(O. E i c h l e r a n d A. F a r a h , ed.) S p i n g e r Verlag, B e r l i n .
Hickman, C. P., J r . , R. A. McNabb, J . S. N e l s o n , D. E . V a n
Breemen, a n d D. C o m f o r t .
1964. E f f e c t o f c o l d a c c l i m a t i o n
on e l e c t r o l y t e d i s t r i b u t i o n i n r a i n b o w t r o u t (Salmo
q a i x d n e r i i ) . Can. J . Z o o l . 42: 577-597.
H i n e g a r d n e r , R.
1969. E v o l u t i o n o f c e l l u l a r DNA c o n t e n t i n
teleost fishes.
Amer. N a t u r . 102: 517-523.
H o c h a c h k a , P. W. a n d G. N. Somero.
1968. The a d a p t a t i o n o f
enzymes t o t e m p e r a t u r e .
Comp. B i o c h e m . P h y s i o l . 2 7 :
659-688.
86
Hochachka,
P. W. a n d J . K. L e w i s .
1970.
In press.
Holmes, R. S. a n d C. L. M a r k e r t .
1969. Immunochemical
h o m o l o g i e s among s u b u n i t s o f t r o u t l a c t a t e d e h y d r o g e n a s e
isozymes.
N a t . A c a d . S c i . , P r o c . 64: 205-210.
Ingram, V. M.
1963. The H a e m o g l o b i n s i n G e n e t i c s a n d
Evolution.
C o l u m b i a U n i v e r s i t y P r e s s , N.Y.
Jankowsky, H.
1960. Uber d i e h o r m o n a l e B e e i n f l u s s u n g d e r
Temperaturadaptation
b e i m G r a s f r o s c h Rana t e m p o r a r i a .
Z. V e r g l . P h y s i o l . 4 3 : 392-410.
J o h n s t o n , P. V. a n d B. I . R o o t s .
1964. B r a i n l i p i d f a t t y
a c i d s a n d t e m p e r a t u r e a c c l i m a t i o n . Comp. B i o c h e m .
P h y s i o l . 1 1 : 303-309.
J o l l e y , W. B., H. W. A l l e n , a n d 0. M. G r i f f i t h .
1967. U l t r a c e n t r i f u g a t i o n u s i n g a c r y l a m i d e g e l . A n a l . Biochem.
21: 454-461.
Kanai,
T. a n d J . S z e r b .
1965. M e s e n c e p h a l i c
reticular
a c t i v a t i n g system and c o r t i c a l a c e t y l c h o l i n e output.
N a t u r e 205: 80-82.
K a r l i n , A.
1967. C h e m i c a l d i s t i n c t i o n s b e t w e e n a c e t y l c h o l i n e s t e r a s e and the a c e t y l c h o l i n e r e c e p t o r .
Biochim.
B i o p h y s . A c t a . 139: 358-362.
Kim,
N.. C , A. D ' l o r i o , a n d W. K. P a i k .
1966. The a c t i v i t y
o f LDH i s o z y m e s d u r i n g t h y r o x i n e i n d u c e d t a d p o l e
metamorphosis.
Can. J . B i o c h e m . 4 4 : 303-310.
K o e l l e , G. B.
1969. S i g n i f i c a n c e o f a c e t y l c h o l i n e s t e r a s e i n
central synaptic transmission.
F e d e r a t i o n P r o c . 28:
95-100.
K o n i s h i , J . a n d C. P. Hickman, J r . 1964. T e m p e r a t u r e
a c c l i m a t i o n i n the c e n t r a l nervous system o f rainbow
t r o u t (Salmo g a i r d n e r i i ) .
Comp. B i o c h e m . P h y s i o l . 13:
433-442.
K o s t e r , . J . F . a n d C. V e e g e r .
1968. The r e l a t i o n b e t w e e n
temperature-inducible a l l o s t e r i c e f f e c t s and the a c t i v a t i o n
energies o f amino-acid oxidases.
Biochim.
Biophys.
A c t a 167: 48-63.
K r n j e v i c , K.
1969. C e n t r a l c h o l i n e r g i c
P r o c . 28: 113-120.
pathways.
Federation
K u s a k i n a , A. A.
1963. R e l a t i o n o f m u s c l e a n d c h o l i n e s t e r a s e
thermostability i n certain fishes to specific
environmental temperature c o n d i t i o n s .
Federation
P r o c . 2 2 : T123-126.
87
Kwon, T. W. a n d H. S. O l c o t t .
1965. Tuna m u s c l e a l d o l a s e I.
P u r i f i c a t i o n and p r o p e r t i e s .
Comp. B i o c h e m . P h y s i o l .
15: 7-16.
L e u z i n g e r , W., M. G o l d b e r g , a n d E . C a u v i n .
1969. M o l e c u l a r
properties of acetylcholinesterase.
J . Mol. B i o l .
4 0 : 217-225.
Licht,
P.
1967. T h e r m a l a d a p t a t i o n i n t h e enzymes o f l i z a r d s
i n r e l a t i o n t o p r e f e r r e d b o d y t e m p e r a t u r e s , p . 131-145.
I n " M o l e c u l a r Mechanisms o f T e m p e r a t u r e A d a p t a t i o n . "
(C. L . P r o s s e r , ed.) Amer. A s s . A d v a n c e . S c i . P u b l .
No. 84, H o r n - S h a f e r , B a l t i m o r e .
Lowry, 0. H., N. J . R o s e n b r o u g h , A. L . F a r r , a n d R. J . R a n d a l l .
19 51.
P r o t e i n measurement w i t h t h e f o l i n p h e n o l
reagent.
J . B i o l . Chem. 193: 265-275.
McKearns, K. W.
1963. Hormonal r e g u l a t i o n o f d e h y d r o g e n a s e
enzymes o f t h e a n t e r i o r p i t u i t a r y .
Biochim. Biophys.
A c t a 73: 507-509.
M a i n , A. R.
1969. K i n e t i c e v i d e n c e o f m u l t i p l e r e v e r s i b l e
c h o l i n e s t e r a s e s based on i n h i b i t i o n b y organophosphates.
J . B i o l . Chem. 244: 829-840.
M a r k e r t , C. L . a n d R. L . H u n t e r .
e s t e r a s e s i n mouse t i s s u e s .
7: 42-49.
1959. The d i s t r i b u t i o n o f
J . H i s t o c h e m . Cytochem.
M a r t i n , R. G. a n d B. N. Ames.
1961. A method f o r d e t e r m i n i n g
the s e d i m e n t a t i o n b e h a v i o u r o f enzymes:
Application to
protein mixtures.
J . B i o l . Chem. 2 3 6 : 1372-1379.
M a r t i n , R. a n d P. R o s e n b e r g .
1968. F i n e s t r u c t u r a l a l t e r a t i o n s
a s s o c i a t e d w i t h venom a c t i o n on s q u i d g i a n t n e r v e f i b e r s .
J . C e l l . B i o l . 36: 341-353.
M a s s a r o , E . J . a n d C. L . M a r k e r t .
1968. Isoenzyme p a t t e r n s o f
salmonid f i s h e s :
Evidence f o r m u l t i p l e c i s t r o n s f o r
l a c t a t e dehydrogenase p o l y o p e p t i d e s .
J . Exp. Z o o l .
168: 223-238.
Massey, V., B. C u r t i , a n d H. G a n t h e r .
1966. A t e m p e r a t u r e d e p e n d e n t c o n f o r m a t i o n a l change i n D-amino a c i d o x i d a s e
a n d i t s e f f e c t on c a t a l y s i s .
J . B i o l . Chem. 2 4 1 :
2347-2357.
M e t z e n b e r g , R. L., M. M a r s h a l l , W. K. P a i k , a n d P. P. Cohen.
1961.
The s y n t h e s i s o f c a r b a m o y l p h o s p h a t e s y n t h e t a s e
i n thyroxine treated tadpoles.
J . B i o l . Chem. 236:
162-165.
M o r t o n , R. K.
1955. Methods o f e x t r a c t i o n o f enzymes f r o m
a n i m a l t i s s u e s , p. 25-51.
I n "Methods i n E n z y m o l o g y . "
V o l . 1. (S. P. C o l o w i c k a n d N. 0. K a p l a n , e d . ) .
A c a d e m i c P r e s s , N.Y.
88
Nachmansohn, D.
Activity.
1959.
C h e m i c a l and M o l e c u l a r
Academic Press,
N.Y.
Nachmansohn, D.
1967.
La membrane e x c i t a b l e .
liees a la bioelectrogenese.
B u l l . Soc.
49: 1177-1189.
Basis
of
Nerve
Macromolecules
Chim. B i o l .
Nachmansohn, D.
1968.
Proteins i n b i o e l e c t r i c i t y :
o f i o n movements a c r o s s e x c i t a b l e membranes.
S c i . , P r o c . 61: 1034-1041.
The
Nat.
Nachmansohn, D.
1969.
P r o t e i n s o f e x c i t a b l e membranes,
187-224.
I n "Membrane P r o t e i n s . "
N.Y. H e a r t A s s .
L i t t l e , Brown a n d Co.,
Boston.
control
Acad.
p.
Symp.
Namba, T. and D. G r o b .
1969.
C h o l i n e s t e r a s e a c t i v i t y o f the
m o t o r e n d p l a t e i n i s o l a t e d m u s c l e membrane.
J . Neurochem.
15: 1445-1454.
Ohno, S. and N. B. A t k i n .
1966.
C o m p a r a t i v e DNA v a l u e s
chromosome complements o f e i g h t s p e c i e s o f f i s h .
Chromosoma 18: 455-466.
and
Ohno, S., V. W o l f , a n d N. B. A t k i n .
1968.
f i s h t o mammals b y gene d u p l i c a t i o n .
169-187.
Evolution
Hereditas
from
59:
Oki,
Y., M. T a k e d a , and S. N i s h i d a .
1966.
p h y s i o l o g i c a l v a r i a t i o n s of esterases
N a t u r e 212: 1390-1391.
Genetic
and
i n mouse serum.
Orr,
P. R.
1955.
Heat death I I .
e n t i r e a n i m a l (Rana p i p i e n s )
P h y s i o l . Z o o l . 28: 294-302.
D i f f e r e n t i a l response of
and s e v e r a l o r g a n systems.
P a p a h a d j o p o u l o s , D.
1968.
Surface p r o p e r t i e s of a c i d i c
phospholipids:
I n t e r a c t i o n o f m o n o l a y e r s and h y d r a t e d
l i q u i d c r y s t a l s w i t h u n i - and b i - v a l e n t m e t a l i o n s .
B i o c h i m . B i o p h y s . A c t a 163: 240-2 54.
P h i l l i s , J . W.
f r o m the
arousal.
and G. C. Chong.
1965.
Acetylcholine release
cerebellar cortices: Its role i n c o r t i c a l
N a t u r e 207: 1253-1255.
P o d l e s k i , T. R.
1969
M o l e c u l a r f o r c e s a c t i n g between
ammonium i o n s and a c e t y l c h o l i n e r e c e p t o r .
Biochem.
P h a r m a c o l . 18: 211-22 5.
Prosser,
on
Z.
C. L. and E . F a r h i .
1965.
E f f e c t s of temperature
c o n d i t i o n e d r e f l e x e s and on n e r v e c o n d u c t i o n i n f i s h .
V e r g l . P h y s i o l . 50: 91-101.
P r o s s e r , C. L. and T. N a g a i .
1968.
E f f e c t s o f low t e m p e r a t u r e
on c o n d i t i o n i n g i n g o l d f i s h , p. 171-180.
In
"The
C e n t r a l N e r v o u s System a n d F i s h B e h a v i o u r . "
(D. I n g l e ,
ed.)
Univ. Chicago Press, Chicago.
89
Rahn, H.
1965.
Gas t r a n s p o r t f r o m t h e e x t e r n a l e n v i r o n m e n t
t o the c e l l , p. 3-23.
I n "Development o f the L u n g . "
(A. V. S. De Reuck and R. P o r t e r , e d . ) .
C i b a Found.
Symp.
J . and A. C h u r c h i l l L t d . , L o n d o n .
Read,
K. R.
1964.
Comparative b i o c h e m i s t r y of a d a p t a t i o n s
o f p o i k i l o t h e r m s t o t h e t h e r m a l e n v i r o n m e n t , p. 39-47.
I n " P r o c . Symp. Exp. M a r i n e E c o l . "
Occasional Publication,
No. 2.
Graduate S c h o o l o f Oceanography, U n i v . o f
Rhode I s l a n d .
Read, K. R. H.
1967.
T h e r m o s t a b i l i t y of proteins i n
p o i k i l o t h e r m s , p. 93-106.
In "Molecular
Mechanisms
of Temperature A d a p t a t i o n . "
(C. L. P r o s s e r ,
ed.)
Amer. A s s . A d v a n c e . S c i . P u b l . No. 84, H o r n - S h a f e r ,
Baltimore.
Reeves, R. B. a n d T. L. W i l s o n .
1969.
I n t r a c e l l u l a r pH i n
• b u l l f r o g s t r i a t e d m u s c l e and c a r d i a c m u s c l e as a
f u n c t i o n of body temperature.
F e d e r a t i o n P r o c . 28:
782.(Abstr.)
R o d r i g u e z De L o r e s A r n a i z , G., M. A l b e r i c i and E . De R o b e r t i s .
1967.
U l t r a s t r u c t u r e and e n z y m i c s t u d i e s on c h o l i n e r g i c
a n d n o n - c h o l i n e r g i c s y n a p t i c membranes i s o l a t e d f r o m b r a i n
cortex.
J . Neurochem. 14: 215-22 5.
Roots,
B. I .
1968.
Phospholipids of g o l d f i s h (Carassius
a u r a t u s L.) b r a i n :
The i n f l u e n c e o f e n v i r o n m e n t a l
temperature.
Comp. B i o c h e m . P h y s i o l . 25: 457-466.
Roots,
B. I . and C. L. P r o s s e r .
1962.
and t h e n e r v o u s s y s t e m i n f i s h .
629*
Temperature a c c l i m a t i o n
J . Exp. B i o l . 39:
617-
%
R o s e n b e r g , P.
Toxicon
1965.
E f f e c t s o f venoms on
3: 125-131.
R o s e n z w e i g , M. R.
1957.
La c h i m i e
individual.
(Brain chemistry
P s y c h o l . F r a n c . 1: 10-11.
the
squid
du . c e r v e a u e t
and i n d i v i d u a l
axon.
l a comporte
behaviour.)
R u s s e l l , R.
W.
1969.
Behavioural aspects of c h o l i n e r g i c
transmission.
F e d e r a t i o n P r o c . 28: 121-131.
R u t t e r , W. J .
1964.
23: 1248-12 57.
E v o l u t i o n of a l d o l a s e .
Federation
Proc.
S c h l a e p p e r , W. W. and R. M. T o r a c k .
1966.
The u l t r a s t r u c t u r a l
l o c a l i z a t i o n o f c h o l i n e s t e r a s e a c t i v i t y i n the s c i a t i c
nerve o f the r a t .
J . H i s t o c h e m . C y t o c h e m . 14: 369-378.
90
S e k u z u , I . , P. J u r t s h u k , J r . , and D. E . G r e e n .
1963.
Studies
on the e l e c t r o n t r a n s f e r s y s t e m .
L i . i s o l a t i o n and
c h a r a c t e r i z a t i o n o f the D - ( - ) - h y d r o x y b u t y r i c apodehydrogenase
from beef h e a r t m i t o c h o n d r i a .
J . B i o l . Chem. 238:
975982.
S i l m a n , H. I . and A. K a r l i n .
1967.
Effect
c a u s e d b y s u b s t r a t e h y d r o l y s i s on t h e
membrane b o u n d a c e t y l c h o l i n e s t e r a s e .
P r o c . 58: 1664-1668.
S m i t h i e s , 0., G. E . C o n n e l l ,
r e a r r a n g e m e n t s and the
N a t u r e 196: 232-236.
o f l o c a l pH c h a n g e s
a c t i v i t y of
Nat. Acad. S c i . ,
and G. H. D i x o n .
1962.
Chromosomal
e v o l u t i o n of h a p t o g l o b i n genes.
Somero, G. N.
1969.
E n z y m i c mechanisms o f t e m p e r a t u r e
compensation:
Immediate and e v o l u t i o n a r y e f f e c t s o f
t e m p e r a t u r e on enzymes o f a q u a t i c p o i k i l o t h e r m s .
Amer.
N a t u r . 103: 517-530.
Somero, G. N. and A. L. D e V r i e s .
o f some a n t a r c t i c f i s h e s .
1967.
Science
Temperature t o l e r a n c e
156: 257-258.
Somero, G. N.
and P. W. H o c h a c h k a .
1968.
The e f f e c t o f
t e m p e r a t u r e on c a t a l y t i c and r e g u l a t o r y f u n c t i o n s o f
p y r u v a t e k i n a s e s o f the r a i n b o w t r o u t and the a n t a r c t i c
f i s h , Trematomus b e r n a c c h i i .
B i o c h e m . J . 110: 395-400.
Somero, G. N. a n d P. W. H o c h a c h k a .
1969.
Isoenzymes and
s h o r t term temperature compensation i n p o i k i l o t h e r m s :
A c t i v a t i o n of l a c t a t e dehydrogenase isoenzymes by
temperature decreases.
N a t u r e 223: 194-195.
Srere,
P. A.
1968.
S t u d i e s on p u r i f i e d c i t r a t e enzymes:
M e t a b o l i c i n t e r p r e t a t i o n s , p. 11-21.
In "Metabolic
Roles o f C i t r a t e . "
(T. W. Goodwin, e d . ) .
Biochem.
Symp. No. 27.
Academic Press, London.
Soc.
Stadtman, E. R.
1966.
A l l o s t e r i c r e g u l a t i o n o f enzyme a c t i v i t y
p. 41-154.
I n "Advan. E n z y m o l . " ( F . F. Nord, e d . ) .
I n t e r s c i e n c e P u b l i s h e r s , N. Y.
T a n a k a , R., and K. P. S t r i c k l a n d . 1965.
Role of p h o s p h o l i p i d
i n the a c t i v a t i o n o f N a ,
K
- a c t i v a t e d adenosine
t r i p h o s p h a t a s e of b e e f b r a i n . A r c h . Biochem. B i o p h y s .
I l l : 583-592.
+
+
T o b i a s , J . M., D. P. A g i n , and R. P a l o w s k i .
1962.
P h o s p h o l i p i d - c h o l e s t e r o l membrane m o d e l : c o n t r o l o f
r e s i s t a n c e by ions or c u r r e n t flow.
J . Gen. P h y s i o l .
45: 989-1001.
Tomkins, G. M., T. D. G e l e h r t e r , D. G r a n n e r , D. M a r t i n , J r . ,
H. H. Samuels and E. B. Thompson.
1969.
Control of
s p e c i f i c gene e x p r e s s i o n i n h i g h e r o r g a n i s m s .
Science
166: 1474-1480.
91
T z a g o l o f f , A. a n d D. H. MacLennan.
1965. S t u d i e s o f t h e
e l e c t r o n t r a n s p o r t system.
LXIV. Role o f p h o s p h o l i p i d s
i n cytochrome o x i d a s e .
B i o c h i m . B i o p h y s . A c t a 99:
476-485.
Ushakov, B. P.
1967. C o u p l e d e v o l u t i o n a r y c h a n g e s i n p r o t e i n
t h e r m o s t a b i l i t y , p . 107-130.
I n " M o l e c u l a r Mechanisms
of Temperature A c c l i m a t i o n . "
(C. L . P r o s s e r , e d . ) .
Amer. A s s . A d v a n c e . S c i . P u b l . No. 84, H o r n - S h a f e r ,
Baltimore.
V a n a t t a , J . C.
1969. Exchange o f p h o s p h o l i p i d - b o u n d Na i n
toad bladder with
Na.
S o c . E x p . B i o l . Med., P r o c .
131: 1450-1453.
2 4
V a r n e r , J . E . , G. Ramchandra.
1964. H o r m o n a l c o n t r o l o f
enzyme s y n t h e s i s i n b a r l e y e n d o s p e r m .
Nat. Acad. S c i . ,
P r o c . 52: 100-106.
V e g o t s k y , A. a n d E . F r i e d e n ,
1958. The s i g n i f i c a n c e o f
s u b s t r a t e c o n c e n t r a t i o n t o i n v i v o enzyme r e a c t i o n s .
E n z y m o l o g i a 19: 143-150.
V e s s e l l , E . S. a n d K. L . Y i e l d i n g .
1966. E f f e c t o f pH, i o n i c
s t r e n g t h and m e t a b o l i c i n t e r m e d i a t e s on the r a t e s o f
h e a t i n a c t i v a t i o n o f l a c t a t e dehydrogenase
isozymes.
N a t . A c a d . S c i . , P r o c . 56: 1317-1324.
Vroman, H. E . a n d J . R. C. Brown.
1963. E f f e c t o f t e m p e r a t u r e
on t h e a c t i v i t y o f s u c c i n i c d e h y d r o g e n a s e f r o m t h e
l i v e r s o f r a t s and f r o g s .
J . C e l l . Comp. P h y s i o l .
61: 129-131.
Walsh,
R. R. a n d S. E . D e a l .
1957. R e v e r s i b l e c o n d u c t i o n
b l o c k p r o d u c e d b y l i p i d - i n s o l u b l e q u a t e r n a r y ammonium
ions i n cetyl-trimethylammonium bromide t r e a t e d nerves.
Amer. J . P h y s i o l . 197: 547-550.
W a t l i n g t o n , C. 0. a n d W. R. H a r l a n ,
and l i p i d c o n t e n t o f i s o l a t e d
P h y s i o l . 2 1 7 : ' 1004-1008.
J r . 1969.
frog skin.
Ion t r a n s p o r t
Amer. J .
Watts,
D. C.
1968. V a r i a t i o n i n enzyme s t r u c t u r e a n d f u n c t i o n :
The g u i d e l i n e s o f e v o l u t i o n , p . 1-114.
I n "Advances
i n Comparative P h y s i o l o g y and B i o c h e m i s t r y . "
V o l . 3.
(0. L o w e n s t e i n , e d . ) .
A c a d e m i c P r e s s , N.Y.
Watts,
R. L . a n d D. C. W a t t s .
1969. Gene d u p l i c a t i o n a n d t h e
e v o l u t i o n o f enzymes.
N a t u r e 217: 112 5-1130.
Weiss,
B. a n d A. H e l l e r .
1969. M e t h o d o l o g i c a l p r o b l e m s i n
e v a l u a t i n g t h e r o l e o f c h o l i n e r g i c mechanisms i n
behaviour.
F e d e r a t i o n P r o c . 2 8 : 135-146.
92
W e t l a u f e r , D. B.
1962. U l t r a v i o l e t s p e c t r a o f p r o t e i n s a n d
amino a c i s , p. 303-390.
I n "Advances i n P r o t e i n C h e m i s t r y . "
V o l . 17. (C. B. A n f i n s e n , J r . , M. L . Ansohn, K. B a i l e y ,
a n d J . T. E d s a l l , e d . ) . A c a d e m i c P r e s s , N.Y.
W h i t t a k e r , V. P.
1965. The a p p l i c a t i o n o f s u b c e l l u l a r
f r a c t i o n a t i o n techniques t o the study o f b r a i n f u n c t i o n .
P r o g . B i o p h y s . M o l . B i o l . 15: 39-96.
W i l s o n , I . B. a n d E . C a b i b .
1956.
Acetylcholinesterase:
E n t h a l p i e s and e n t r o p i e s o f a c t i v a t i o n .
J . Amer. Chem.
Soc. 78: 202-207.
W i l s o n , I . B. a n d M. Cohen.
1953. The e s s e n t i a l i t y o f
acetylcholinesterase i n conduction.
Biochim. Biophys.
A c t a . 11: 147-156.
Y a n a r i , S. a n d F . A. B o v e y .
1960. I n t e r p r e t a t i o n o f t h e
u l t r a v i o l e t s p e c t r a l changes o f p r o t e i n s .
J . B i o l . Chem.
235: 2818-2826.