Journal
.T comn.
Phvsiol.
119.195-206
(1977)
of
Comparative
Physiology.
(C) by Springer-
B
V erlag
1977
Molecular Mechanisms of Temperature Adaptation
in Fish Myofibrillar Adenosine Triphosphatases
Ian
A.
Johnstonl
and
N.J.
Walesby2
1 Department of Physiology, University of St. Andrews, St. Andrews, Fife, Scotland
2 Life Sciences Division. British Antarctic Survey, Madingley Road, Cambridge, England
Received January 5. 1977
Summary. Studies have been carried out on the Mg2+Ca2+-myofibrillar
A TPase from the muscles of fish adapted to different environmental temperatures. The thermal stability of the A TPase is strongly correlated with mean
habitat temperature. Activities of Antarctic fish A TPases are significantly
higher at low temperatures than those of temperate and tropical water
species. The effects of ionic strength on A TPase activity have also been
studied. The Gibbs free energy of activation (JG*) was found to increase
and enzyme activity decrease with increasing ionic strength within the physiological temperature range of each species. Significantly lower values of
JG*, of around 1 Kcal/mole, are obtained for the A TPase of cold-adapted
compared to tropical fish. Enthalpic and en tropic activation energies were
also reduced in the cold adapted A TPases. It is postulated that the reduction
of the enthalpic activation term in the cold adapted enzyme confers the
advantage of reducing the temperature sensitivity of the rate limiting step
thus partly compensating for the low heat content of the ce.1lular environment.
Possible molecular mechanisms of temperature compensation in fish myofihrillar A TPases are discussed.
Introduction
Poikilothermic vertebrates successfully exploit thermal environments in the range
-2 °C to 40 °C. Many species show adaptive changes which allow them to exhibit
similar rates of physiological activity in spite of widely different body temperatures. Evolutionary adaptation to relatively narrow temperature ranges has
resulted in the selection of homologous enzymes each with characteristic physical
and catalytic properties (Hazel and Prosser, 1974). Interpretation of comparative
studies on the thermodynamic and kinetic properties of poikilothermic enzymes
is often made difficult by the absence of reliable kinetic models. In the case
of muscle contraction, a kinetic scheme for the hydrolysis of A TP by myosin
subfragment 1 in dilute solution has been elucidated which involves at least
.
.A. Johnston
196
and N.J.
Walesby
7 intermediate steps (Trentham et al., 1976). Correspondingly detailed kinetic
schemes relating to crossbridge cycling mechanisms and actin-myosin interactions in the intact myofibrillar matrix are not currently available for any system
(Hill and Eisenberg, 1976). The uncertainties and complexities of kinetic modelling have confmed most comparative studies to determinations of catalytic rate
constants (KcaJ of activation (Bendall, 1969; Low et al., 1973; Johnston and
Goldspink, 1975). Although direct mechanistic interpretations of such studies
are difficult, they have nevertheless proved useful in establishing a framework
for investigating some of the underlying strategies of temperature adaptation
in poikilotherms (Hochachka and Somero, 1973; Precht et al., 1973; Hazel
and Prosser, 1974).
In the case of fast twitch muscles of fish, the A TPase activity of myofibrils
from cold adapted species is considerably higher at low temperatures than
for tropical species (Johnston et al., 1975a). Differences in rate compensation
between cold and warm adapted A TPases are reflected in changes in thermodynamic activation parameters (Johnston and Goldspink, 1975). In particular,
the relative contributions of enthalpic and entropic activation terms to the
free energy of activation varies according to environmental temperature. In
a preliminary study it was concluded that reduction of the enthalpy term in
the myofibrillar A TPase from cold-adapted muscles may confer an energetic
advantage in reducing the temperature sensitivity of the activation process (Johnston and Goldspink, 1975).
Small reductions in Gibbs free energy of activation for cold-adapted A TPases
might also result in significant increases in reaction rate at low temperatures
(cf. Low et al., 1973). In the present study activation parameters for the
Mg2 +Ca 2+-activated A TPase have been compared for fish inhabiting different
environmental temperatures. Since the A TPase activity is highly sensitive to
ionic strength, the effect of this parameter on the rate and thermodynamic
activation terms has also been investigated.
Materials and Methods
Fi.h
Two species of Antarctic fish were used in these investigations. Notothenia neglecta were caught
by trammel net at Signy Island, South Orkney Islands. The single specimen of haemoglobinless
"ice-fish" (Chaenichthyiidae),
Champsocephalus gunnari, was obtained by otter trawl from 250 m
depth off South Georgia, and was the first specimen of any ice-fish to be kept alive outside
Antarctica. Fish were transported to the U.K. in tanks of filtered recirculated seawater maintained
at loC. Indian Ocean species were obtained from local fish dealers and kept in aquaria at their
habitat temperature (26 :t I °C) for several weeks before use. Specimens of the North Sea fish
CoItus bubalis L. were obtained from local fishermen at Pittenweem, East Fife, during November
and maintained
in tanks of recirculated
seawater at 4 °C.
Preparation of Myo{ibrils
Fish were stunned by a blow to the head and killed by decapitation. Several grams of white
epaxial muscle were immediately dissected from the trunk. The muscle was minced with scissors
and homo17"n;""t1 "t ()Or with"
P...lvtr...n hlpnt!pr f...r .'vM\.
;n 01 M11"rl
10~~A T..;oU£"'1
-
Temperature
Adaptation
in Fish Myofibrillar
A TPases
107
buffer at pH 7.0. The extent of homogenisation was monitored by microscopical examination.
The homogenate was centrifuged at 10,000 x 9 for 10 min and the myofibrils prepared from the
residue by the method of Perry and Qrey (1956). In preliminary experiments following preparation
myofibrils were treated with a 1% solution of Triton X-I00 as described by Solaro et al. (1971).
Treatment with Triton solubilises the sarcoplasmic reticulum and reduces possible contamination
with membranous A TPases without affecting the myofibrillar A TPase activity (Solaro et al., 1971).
The detergent was removed by washing 5 times in 50 vols. of 0.1 M KCI, 10 mM Tris-HCI, pH 7.0.
The contribution
of sarcoplasmic reticulum A TPases to the total measured activity in the
original preparation was found to be negligible. Myofibrils
were also tested for contamination
by mitochondrial ATPases as previously described (Johnston and Tota, 1974). Preparations were
essentially free of non-myofibrillar
A TPases under the assay conditions employed.
Myofibrils were finally suspended in the preparation medium at a concentration of approximately
10 mg/ml. Protein concentration was determined by a standardised biuret method (Gornall et al.,
1949).
Enzyme Assay
The standard assay for Mg2+Ca2+-activated
myofibrillar
ATPase was performed in a volume
of I ml of 50 mM Tris-HCI pH 7.5,5 mM disodium ATP, 5 mM MgCI2, 0.1 mM CaCI2 at a myofibril concentration of 0.4-0.5 mg/ml and ionic strength of 0.10 (adjusted with KCI). Although
the Mg2 + -activated A TPase activity is being studied trace quantities of calcium are required to
overcome inhibition by the calcium regulatory proteins of the tropomyosin-troponins
complex.
Following Fuchs et al. (1975) we have used the designation Mg2+Ca2+-stimulated
myofibrillar
A TPase since calcium ions in low concentration have been found to reduce the activation enthalpy
at temperatures greater than 15-20°C. The measured A TPase activity therefore closely parallels
the in vivo physiological myofibrillar
A TPase. The reaction was started by addition of A TP to
preincubated myofibrils and terminated with I ml of 10% (w/v) trichloracetic acid. Precipitated
protein was removed by centrifugation
and inorganic phosphate measured in an aliquot of the
supernatant by the method of Rockstein and Herron (1951). Appropriate
enzyme and reagent
blanks were included in all experiments. Preparations were tested for calcium sensitivity in assays
in which 4 mM EGTA replaced added calcium (0.1 mM). Under these conditions the activity was
usuallv less than 10% of that in the Dresence of trace amounts of calcium.
Ionic StrenJ!th Exoeriments
Assay conditions for ionic strength experiments were as follows: 25 mM Tris-HCI
pH 7.5,
5 mM MgCI2, 5 mM ATP, 0.1 mM CaCI2, 0.5 mg/ml myofibrils with the remainder of the ionic
strength (11) being contributed by sodium-p-glycerophosphate,
this being used in preference to
KCI or NaCI since chloride ions have been shown to have an inhibitory effect on the A TPase
activity at very low ionic strengths (11= 0.1) (Bendall, 1964).
Therma/ Inactivation
Experiments
Thermal inactivation
of the Mg2+Ca2+-activated
ATPase was carried out in a w1tter-jacketed
reaction vessel fitted with a magnetic stirrer in a medium of 0.05 M KCI, 40 mM Tris-HCI at
pH 7.5. Myofibrils (0.8-1.0 mg/ml) were added to an 18-fold excess of medium previously incubated
to 37°C. An initial sample was taken within 3 sec and subsequently at appropriate intervals and
pipet ted into tubes cooled in melting ice to prevent further inactivation. Myofibrils partially inactivated by exposure to high temperatures were assayed for ATPase activity at 18°C.
Ca[culation or Thermodvnamic Parameters
Mg2+Ca2+-activated ATPase activity was measured in duplicate at a series of temperatures. Arrhe.
nius plots of fish Mg2+-myofibrillar
ATPase often show a transition break at 15-18°C (Bendall,
I.A. Johnston and N
IQR
Wale.hv
1969; Johnston et al., 1973). In addition, the Mg2+Ca2+-ATPase of cold-adapted myofibrils shows
an initial activation and subsequent rapid thermal denaturation at temperatures in excess of 28-29 oC
(Johnston et al., 1973, 1975a). For this reason measurements of ATPase activity were made for
the temperature ranges 0-15°C and 18-28°C. Apparent activation energies (AH*+RT)
over these
temperature ranges were calculated from the slopes of the corresponding Arrhenius plots.
Thermodynamic activation parameters were calculated by the following relationships as described by Lehrer and Barker (1970):
AG* = AH* -TAS*
AH*=E
. -RT
AS* =4.576 r1ogto k-10.753-logtnT+-",
1:..
.:
4.5761..
The rate constant k (s- I) is proportional
to v max and expressed as moles A TP split/mole
enzyme active site/second.
The proportion
of myosin in the myofibril was assumed to be 54% (Bendall, 1969) with
a molecular mass of 240,000 daltons per enzyme site (Lowey et al., 1969; Godfrey and Harrington,
1970). Statistical analyses were carried out using analvses of variance for eQual sample numbers.
Results and Discussion
Thermal Stabilitv
A measure of the thermal stability of the active site was obtained by assessing
the loss of A TPase activity at 37 oC. In some cases the inactivation of the
Mg2+Ca2+-activated ATPase followed first-order reaction kinetics (Fig. la, b,
c). However, in most warm-adapted enzymes there was an initial increase in
activity following preincubation at 37°C (Fig. le). It would appear that the
initial stages of thermal denaturation result in a conformation which is more
active than the native enzyme. Similar transient increases in activity resulting
from unfolding of the native enzyme have been observed in fish myosins following denaturation by urea and in the thermal denaturation of sarcoplasmic reticulum Ca 2 +-A TPase activities (Syrovy et al., 1970; Carvalho and Santos, 1976).
The half-life of inactivation of the Mg2+Ca2+-activated myofibrillar
ATPase
was positively correlated with adaptation temperature. Three Antarctic species
investigated all showed a half-life of inactivation of less than 1.5 min. The
order of stability Notothenia rossii > Champsocepha/us gunnari > Notothenia
neg/ecta was correlated with degrees longitude south, the most labile A TPase
coming from the most southerly species. This compares with .a half-life of
inactivation of around 10 min for North Sea species (environmental temperature
5-12°C) and 60-100 min for Indian Ocean species (environmental temperature
20-25 OC). The variation in "stability"
of the Mg2 +Ca 2 +-A TPase according
to environmental temperature is illustrated in Figure 2. The most stable enzyme
so far investigated is that from Ti/apia grahami a species living at 36-40 oC
in a hot-springs soda-Iake in central Kenya (tl/2 at 37 oC = 520 min) (Johnston
et al., 1973). There have been numerous reports of protein or cell thermostability
being correlated with habitat temperature in poikilotherms (Ushakov, 1964;
HH7el and Prosser. 1974). It has been su~gested that in some cases heat dama~e
.
Temperature
Adaptation
in Fish Myofibrillar
199
A TPases
"
<I:
~
0,5
1,0
1,5
z,o
2,5
...
~
~
z
...
u
~
...
~
10
20
30
40
JNCURA.TlON
TlME
Fig. 1a-f.
The effect of preincubation
50
100 150 200 250
JNCURA.TlON
TIME
at 37°C
on the thermal
denaturation
of the Mg2Ca 2+
activated myofibrillar
ATPase of fish from different environmental temperatures (ET). Insets are
plots of loglo percent remaining activity against time of exposure to 37 oC. Assay conditions are
given in the text. a Icefish (Champsocephalus gunnari Lonnberg) (Antarctic species; mean ET
-1°C to +2°C). b Notothenia neglecta (Richardson) {Antarctic species; mean ET 0°C to +3°C).
c Saithe (Gadus virens, L.) (North Sea species; mean ET +3°C to +12°C). d Red Mullet (Mullus
barbatus) (Mediterranean species; mean ET +10°C to +21°C). e Amphiprion sebae (Indo-Pacific
species; mean ET + 18 oC to + 26 OC). r Tilapia grahami (Equatorial hotsprings sodalake species Lake Magardi, Kenya; mean ET +35°C
and unpublished results)
to +38°C).
(Taken from Johnston and Goldspink,
1973,
and cell death are limited by the heat denaturation of the least resistant protein
complex. For example, the temperature at which muscle excitability was lost
for 17 species of fish has been correlated with the temperature for inactivation
of cholinesterase activity (Kusakina, 1963). It is far from certain whether or
not such correlations between tissue viability and protein thermostability are
causal. However, it is clear from the present study that change~ in the thermal
~tahilitv of mvofihrillar A TPa~e hetween ~necies adanted to different temDera-
,00
A
Tnhnston
and
N.J.
Walesbv
~
1..
z
o
~
;=
...
..
~
<
~
~
~
1-
~
~
:;'
:
--.i.ANTARCTIC NORTH MEDITERRANEAN
;---.
INOIAN EAST
AFRICAN
EAST
AFRICAN
(X:fAN
SEA
SEA
(X:fAN
LAKE
~NG
.
Fig. 2. The relationship between the half-life of thermal denaturation (minutes) of the Mg2+Ca2+activated myofibrillar
A TPase and environmental
temperature for 27 species of teleosts. Assay
conditions are given in the text. I, Champsocephalus gunnari; 2, Notothenia neglecta; 3, Notothenia
rossii; 4, Gadus virens. ; 5, Myxoxocephalus scorpius. ; 6, Gadus morhua. ; 7, Limanda limanda. ;
8, CoItus bubalis; 9, Pleuronectes platessa. ; 10, Serranus cabri/1a. ; 11, Gobius pagne/1us. ; 12, Diplodus
annularis. ; 13, Mu/Ius barbatus. ; 14, Lithognathus morymus. ; 15, Syngnathus acus. ; 16, Coris
julius. ; 17, Mugil cephalus. ; 18, Abudefduf parafema ; 19, Epinephalus garratophalus. ; 20, Chromis
chromis.; 21, Dascy/1us aruanus; 22, Grammistes sexlineatus.; 23, Amphiprion percula; 24, Halichoeres centriquadras. ; 25, Balistoides spp. ; 26, Tilapia nigra. ; 27, Tilapia grahami.. .data taken
from Johnston et al. 0973)
tures are related to adaptive modifications in the catalytic efficiencies of the
enzymes. These differences in the rates of thermal inactivation between warm
and cold adapted myofibrillar A TPases presumably reflect genetic differences
in protein primary structure. It would appear that selective modifications in
the active site for function at low temperatures have resulted in weaker bonding
in the tertiary structure of the molecule resulting in an A TPase which is thermolabile at high temperatures (Fig. 2).
Rate and Thermndvnamir
Activl1finn
Parnmpfpr.~
Thermodynamic activation parameters for the Mg2 +Ca 2 +-activated myofibrillar
A TPase are given in Tables 1 and 2. Maximal reaction rates of the Antarctic
fish A TPase are significantly higher at low temperatures than those of the
temperate and tropical water species (P<O.OOl). Rate compensation at low
temperatures in the cold adapted A TPase is reflected in lower enthalpies and
entropies of activation, thus confirming earlier studies (Johnston and Goldspink,
.
201
Temperature Adaptation
in Fish Myofibrillar
Table I. Thermodynamic
activation parameters of the Mg2 + Ca 2+ -myofibrillar
at different environmental
Species
A TPases
A TPase of fish living
temperatures. Calculated for 0°C; values are means :tS.E.
Environmental
N
temperature
v ma" x10-2
AH*
AS*
AO..
at
(c31(mole)
(e.u.)
(cal/mole}
00 C
(I!moles
.mg-l.min-l)
Antarctica
-I oC to + 2 O(
9
7.70:tO.30
7,400:t230
-31.04:tO.79
IS,870:t20
Notothenia
neg/ecta
Antarctica
0°C to +3°C
Q
4.80 :t 0.20
11,300:!: 160
-17.76:!:0.63
16,130:!:20
CoItus
buba/is
North Sea
3 oc to 12°C
Q
3.50:tO.O8
13,550:tllO
-9.86:tO.43
16,290:t12
Dascyllus
carneus
Indo-Pacific
18°C to 26°C
9
0.37 :t 0.046
25,350 :t 980
+ 25.08 :t4.04
17,540 :t 70
Pomatocentrus
uniocellatus
Indo-Pacific
18°C to 26°C
9
0.31 :!:0.03
26,500 :t 850
+ 32.47 :t 3.03
17 ,620 :t 50
Ice-fish
( Champsocepha/us
gunnarl)
Table 2. A comparison of the thermodynamic activation parameters of a tropical and Antarctic
Mg2 + Ca2 + -myofibrillar
A TPase at their respective physiological temperatures
Assayed temperature
range"
Species
0-15 °C
Antarctic
(Champsocephalus
gunnari)
0-15 °C
Tropical
(Pomatocentrus
a
18-28 °C
uniocellatus)
Thermodynamic
18'-28 °C
AH+
AS*
,10*
(cal/mole)
(e.u.)
(cal{mole)
8,040
7,990
-27.39
-27.55
28,000
24,500
parameters calculated from mean of two preparations
and 26 °C for lower and upper temperature
fish
37.39
25.39
15,520
16,220
17,74(1
16,900
for temperatures 0°C
ranges, respectively
1975). The net entropy of activation varies from negative values for cold to
high positive values for tropical adapted A TPases. Lower LIH* and LIS* values
can be attributed to the breaking of fewer weaker bonds during the dissociation,
or the making of more bonds during the formation, of the activated complex.
Similar differences in the relative contributions of LlH* and LIS* for muscle
lactate dehydrogenase and glycogen phosphorylase between poikilotherms and
homeotherms have been attributed to differences in the numbers of weak bonds
formed during the activation process (Low and Somero, 1974). In either case,
reduction of the enthalpic contribution to LlG* might be expected to confer
an energetic advantage to the cold-adapted enzyme by reducing the temperature
dependence of the activation process. Further rate compensation at low temperatures in the Antarctic A TPase is probably achieved through a reduction in
Gibbs free energy of activation. The free energy of activation for the Antarctic
fish Mg2+Ca2+-ATPase is around 1.500 cal.mole-l
lower than that for the
.
=
.
~
~
c
~
~
=
;;:
c
>
~
~=
~
.t.
u
=0
3,4
3,5
3,6
3,7
I/T"K x 103
Fig. 3. Arrhenius p!ot of )oglo Mg2+Ca2+-activated
ATPase activity (moles ATP split.mole ,
myosin active site- I. s- ') against liT (OK x !03) for white muscle myofibrils of the icefish, Champ- .
socephalus gunnari (ET -! oC to +2°C).
text. Significance of regression P<O.OO!
Mean of two preparations.
Assay conditions
given in
m
~
~
0«
a:
~
~
~
..:
0
>
~
=
~
~
u
~
0
3,3
3,4
3.5
l/r'K
3,6
3.1
x 103
Fig, 4, Arrhenius plot of log10 Mg2+-Ca2+-activated
ATPase activity (moles ATP split.mole
myosin active site- I.S- 1) against I/T (OK x 103) for white muscle myofibrils of an Indo-Pacific
species Pomatocentrus uniocellatus (ET + 18 °C to + 26 °C). Mean of two preparations. Assay
conditions given in text. Regressions over the ranges o°C to + 15 °C and + 18 °C to +28 °C
sil!nificant at the P=O.OI level
.
"'"--
Temperature Adaptation
in Fish Myofibrillar
A TPases
203
tropical species (P < 0.001; Tables 1 and 2). This will have the effect of increasing
the proportion of molecules with sufficient energy to form the activated complex
at any given temperature. Arrhenius plots for the Antarctic species Champsocephalus gunnari were linear in the range 0-28°C (P<0.001) (Fig.3). In contrast,
the Indian Ocean species Pomatocentrus uniocellatus showed a transition break
in temperature dependency at around 15-18°C (Fig.4; Table 2). Non-linear
Arrhenius plots of rabbit actomyosin A TPase have been attributed to the binding
of Ca 2+ to its receptol site on the regulatory protein troponin C (Hartshorne
et al., 1972). Preparations of synthetic actomyosin which did not contain protein
of the tropomyosin-troponinscomplex
were found to have a linear temperature
dependence regardless of the presence or absence of trace amounts of calcium
ions (Hartshorne et al., 1972). However, the molecular mechanism responsible
for non-linear Arrhenius plots of myofibrils may differ between animal groups
since non-linear plots were obtained for both the Mg2+-activated and the
M~2+Ca2+-activatedATPases
of frog skeletal muscle fibrils (Fuchs et al., 1975).
Effect
Qf Ionic Strenf[th
Ionic strength has a marked effect on the temperature dependence of the
Mg2 +Ca 2+-activated myofibrillar A TPase. In both the Antarctic species Champs#)cephalusgunnari and tropical species Dascyllus carneus A TPase activity increased up to an ionic strength of approximately ~= 0.7 and then decreased
with increasing ionic strength at all temperatures studied (Fig. 5). Interestingly,
.
:.
1..
~
~
=
=
;;:
0
,..
~
.t.Q
~
~
IONIC STRENGTH
11'1
Fig.5. A graph showing the effect of ionic strength on Mg2+ Ca2+-myofibrillar
A TPase activity
of Champsocephalus gunnari (Antarctic species) o and Dascyllus carneus (Indo-Pacific species) ..
Assav conditions are I!iven in the text
.
704
I.A. Johnston and N.J. Walesbv
(bl
".
005
015
IONIC STRENGTH (II)
""
--
..
005
""
KJNIC STRENGTH (III
Fig. 6a and b. A graph showing the effect of ionic strength on a the enthalpies of activation
(AH*), and b the free energy of activation (AG*) of the Mg2+Ca2+-ATPase
of white milscle
myofibrils from an Antarctic fish, Champsocephalus gunnari o and an Indo-Pacific fish, Dascyllus
carneus ..Assay conditions are given in the text
the tension developed by skinned muscle fibres is similarly affected by changes
in ionic strength (Barouch and Moos, 1971). The mechanism for the inhibition
of A TPase activity with increasing ionic strength in myofibrillar preparations
is unclear. However, the decrease of acto-heavy meromyosin (HMM) ATPase
with increasing salt concentration has been attributed to decreased actin-myosin
interaction (Barouch and Moos, 1971). At the physiological temperature of
each species AG* increased with ionic strength for both fish studied so that
the free energy of activation of the Antarctic fish was always lower than that
for the tropical species (Fig. 6).
Significance of Temperature Compensation Phenomena
Plots of enthalpy versus entropy change for a wide range of. water-solute interactions are linear with slopes in the region of 280-320°K (called proportionality
constants or compensation temperatures) (Lumry and Erying, 1954; Lumry
and Rajender, 1970). Similar apparently linear enthalpy-entropy relationships
have been observed for a wide range of protein reactions (see Lumry and
Rajender, 1970). It has been suggested that enthalpy-entropy compensation
might arise from some ubiquitous property of water and be a major physiologically important property of proteins in both equilibrium and rate processes
n 1lmrv :lnd Evrinl!. 1954). According; to this hypothesis water properties have
Temperature
Adaptation
in Fish Mvofibri!lar
A TPases
2(\~
been one of the dominant factors in the evolution of poikilothermic enzyme
systems. Although appealing, this hypothesis is difficult to test statistically when
the range of experimental temperatures is less than 50 °K due to correlation
of errors in estimating values of L1H* and L1S* from Arrhenius or Van t'HofT
plots.
Conclusions
Until a detailed kinetic model becomes available for the myofibrillar A TPase
it is not possible to interpret the measured activation energies in terms of
the formation of any particular intermediate or set of intermediates. However,
it is clear that differences in cell temperature have lead to adaptive modifications
in the steady state myofibrillar A TPase activity between species. All the fish
in the present study are from environments showing relatively little seasonal
thermal variation and as such are unlikely to show much capacity for acclimatory
changes in enzyme phenotype (Hazel and Prosser, 1974). However, in a previous
study it was shown that, in common with many enzymes of intermediary metabolism (Hochachka and Somero, 1973; Hazel and Prosser, 1974), myofibrillar
A TPase from the eurythermal species Carassius auratus L. showed adaptive
; changes in rate parameters during temperature acclimation from 0°C to 26 °C
(Johnston et al., 1975b). In addition the myofibrillar
A TPase from goldfish
acclimated to the lower temperature for several months was significantly more
thermolabile than that of warm acclimated fish (Johnston et al., 1975b).
It has been shown that activation enthalpies of rabbit natural actomyosin
are altered as a consequence of the binding of calcium to troponin C (Hartshorne
et al., 1972). The smallest fragment obtainable from myosin with A TPase activity
is the globular head called subfragment I. At present it is not known whether
the observed catalytic compensations for function at different temperatures arise
simply from structural modifications of the active site in subfragment lor
whether co-operative interactions of other myofibrillar proteins are involved.
Furthermore, it is possible that molecular mechanisms responsible for adaptive
changes in catalytic efficiency over evolutionary time periods are distinct from
those occurring during seasonal acclimation. In either case, the observed catalytic
compensations for function at a particular temperature are presumably mediated
through selective changes in protein primary structure.
This work was supported by a grant from the Science Research Council.
References
Barouch, W. W ., Moos, C. : Effect of temperature on actin activation of heavy meromyosin adenosine
triphosphatase. Biochim. Biophys. Acta. 234, 183-189 (1971)
Bendall, I. R. : The myofibri1lar A TPase of various animals in relation to ionic strength and temperature. In: Biochemistry of muscular contraction (ed. I. Gergely), pp. 87-93. Boston, Mass. :
Little Brown & Co. 1964
Benda1l, I.R. : Muscles, molecules and movement, pp. 51 et seq. London: Heinemann 1969
,.
206
I.A. Johnston and N.J. Walesby
Carvalho, C.A.M., Santos, M.S. V. : Effect of heat treatment on the A TPase activity of various
sarcoplasmic reticulum preparations. Experientia 32, 428-429 (1976)
Fuchs, F., Hartshorne, D.J., Barns, E.M. ; ATPase activity and superprecipitation of skeletal muscle
actomyosin of frog and rabbit: effect of temperature on calcium sensitivity. Comp. Biochem.
Physiol. 51B, 165-170(1975)
Godfrey, J.E., Harrington,
W.F. : Self-association in the myosin system at high ionic strength,
I. Sensitivity of the interaction to pH and ionic environment. Biochemistry 9, 886-893 (1970)
Gornall, A.G., Bardawill, C.S., David, M.M. : Determination of serum proteins by means of the
biuret reaction. J. bioI. Chem. 177,751-766 (1949)
Hartshorne, D.J., Barns, E.M., Parker, L., Fuchs, F. : The effect of temperature on actomyosin.
Biochim. Biophys. Acta. 267, 196-202 {1972)
Hazel, J.R., Prosser, C.L. : Molecular mechanisms of temperature compensation in poikilotherms.
Physiol. Rev. 54, 620-670 (1974)
Hill, T.I., Eisenberg, E.; Reaction free energy surfaces in myosin-actin-ATP
systems. Biochemistry
15, 1629-1635 (1976)
Hochachka, P. W., Somero, G.N. : Strategies of biochemical adaptation. Philadelphia;
Saunders
1973
Johnston, I.A., Davison, W., Goldspink, G.; Adaptations in Mg2+-activated myofibrillar
ATPase
activity induced by temperature acclimation. FEBS Letters 50,293-295 (1975b)
Johnston, I.A., Frearson, N., Goldspink, G. : The effects of environmental
temperature on the
properties of myofibrillar
adenosine triphosphatase from various species of fish. Biochem. J.
133,735-738 (1973)
Johnston, I.A., Go1dspink, G. : Thermodynamic activation parameters of fish myofibrillar
ATPase
enzyme and evolutionary ~daptations to temperature. Nature (Lond.) 257, 620-6220975)
Johnston, I.A., Tota, B. ; Myofibrillar
ATPase in the various red and white muscles of the tunny
(Thunnus thynnus, L.) and the tub gurnard (Trigla lucerna, L.). Comp. Biochem. Physiol. 49B,
; 367-374 (1974)
Johnston, I.A., Walesby, N.J., Davison, W., Goldspink, G. ; Temperature adaptation in myosin
of antarctic fish. Nature (Lond.) 254, 74-75 0975a)
Kusakina, A.A. : Relation of muscle and cholinesterase thermostability
in certain fishes to specific
environmental temperature conditions. Fed. Proc., Transl. 22, T123-T126 0963)
Lehrer, G.M., Barker, R. : Conformational
changes in rabbit muscle adolase. Kinetic studies.
Biochemistry 9, 1533-1539 (1970)
Low, P.S., Bada, J.L., Somero, G.N. : Temperature adaptation of enzymes; roles of the free energy,
the enthalpy and the entropy of activation. Proc. Natl. Acad. Sci. USA 70, 430-432 (1973)
Low, P.S., Somero, G.N.; Temperature adaptation of enzymes: a proposed molecular basis for
the different catalytic efficiencies of enzymes from ectotherms and endotherms. Comp. Biochem.
Physiol. 49B, 307-312 (1974)
Lowey, S., Slayter, H.S., Weeds, A.G., Baker, H. : Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J. Mol. BioI. 42, 1-29{1969)
Lumry, R., Eyring, H. ; Conformation
changes of protein. J. Phys. Chem. 58, 110-120 (1954)
Lumry, R., Rajender, S. : Enthalpy-entropy compensation phenomena in water solutions of proteins
and small molecules: a ubiquitous property of water. Biopolymers 9, 1125-12270970)
Perry, S.V., Grey, T.C. : A study of the effects of substrate concentration and certain relaxing
factors on the magnesium-activated
myofibrillar
adenosine triphosphatase. Biochem. J. 64,
184-192 (1956)
Precht, H., Christophersen, J., Hensel, H., Larcher, W. : Temperature and life. Berlin-HeidelbergNew York; Springer 19!3
Rockstein, M., Herron, P. W. ; Colorimetric determination of inorganic phosphate in microgram
quantities. Anal. Chem. 23, 1500-1501 0951)
Solaro, R.J., Pang, D.C., Briggs, F.N.: The purification of cardiac myofibrils with triton X-100.
Biochim. Biophys. Acta 245, 259-262 (1971)
Syrovy, I., Gaspar-Godfroid,
A., Hamoir, G.: Comparative study of the myosins from red and
white muscles of the carp. Archs. int. Physiol. Biochem. 78,919-934 (1970)
Trentham, D.R., Eccleston, J.F., Bagshaw, C.R. : Kinetic analysis of ATPase mechanisms. Quart.
Rev. Biophys. 9, 217-2810976)
Ushakov, B. : Thermostability
of cells and proteins in poikilotherms.
Physiol. Rev. 44, 518-560
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