35 1
MESIBRANE-ENZYME BINDING PKENOMENA AND MUSCLE GLYCOLYSIS*
H . 0 . HULTIN
University of Massachusetts, Amherst, Mass.
The g r e a t bulk of t h e work i n s t u d i e s of post-mortem muscle has
involved t h e s t r u c t u r a l p r o t e i n s o r t h e connective t i s s u e p r o t e i n s ,
although it i s w e l l recognized that many of t h e changes brought about
i n t h e s e p r o t e i n s a r e influenced s i g n i f i c a n t l y by t h e s o l u b l e enzymes
of t h e muscle c e l l . We have s t u d i e d how t h e a c t i v i t i e s of some of t h e
g l y c o l y t i c enzymes i n chicken muscle a r e a f f e c t e d by i n t e r a c t i o n with
s u b c e l l u l a r p a r t i c u l a t e s t r u c t u r e s w i t h a view t o understanding what
c o n t r o l s t h e functioning of t h e s e enzymes post-mortem.
I s h a l l d i s c u s s two c l a s s e s of enzymes which we have s t u d i e d . One
of t h e s e c l a s s e s i s exemplified by t h e dehyd-rogenases, l a c t a t e dehydrogenase (LDH) and glyceraldehyde-phosphate dehydrogenase (GAPDH )
These
f a l l under t h e category of enzymes which a r e v e r y e a s i l y s o l u b i l i z e d
and have, i n f a c t , been c l a s s i c a l l y considered t o e x i s t as s o l u b l e
e n t i t i e s i n t h e sarcoplasm. The second c l a s s a r e t h o s e r e q u i r i n g more
vigorous treatment t o remove them f r o m t h e p a r t i c u l a t e s t r u c t u r e s and
have been recognized as e x i s t i n g a t l e a s t i n p a r t i n a s s o c i a t i o n with
t h e p a r t i c u l a t e s t r u c t u r e s of t h e c e l l . The proposals that I am p u t t i n g
forward involve r e v e r s i b l e binding and s o l u b i l i z a t i o n of t h e enzymes.
Thus, t h e s e enzymes a r e not i n t r i n s i c p a r t s of membranes nor do t h e y
remain i n t h e p a r t i c u l a t e phase a t a l l t i m e s . These enzymes a r e r e p r e s e n t i v e of t h e " e x t r i n s i c " o r "peripheral" p r o t e i n s of membranes
described i n t h e paper by D r M. Stromer
.
.
.
I n many cases, the.binding-solubilization which t a k e s place may
involve s t r u c t u r e s o t h e r t h a n membranes, f o r example, P e t t e ' s group i n
Germany has shown t h a t a number of t h e g l y c o l y t i c enzymes cetn i n t e r a c t
with p u r i f i e d a c t i n (Arnold and P e t t e , 1968, 1970; Arnold e t a l . , 1971),
and Amberson and h i s coworkers a t t h e Marine Biology Laboratory a t Wood's
Hole, Massachusetts, have demonstrated by zonal e l e c t r o p h o r e t i c techniques
t h a t t h e s o l u b l e p r o t e i n s of t h e muscle c e l l can bind t o myosin and o t h e r
s t r u c t u r a l p r o t e i n s under t h e proper conditions (Amberson and Bauer, 1971).
My suggestions a r e going t o be t e n t a t i v e . They a r e meant more t o
s t i m u l a t e d i s c u s s i o n and serve as a b a s i s f o r f u r t h e r experimentation
than as a f i n a l pronouncement on t h e exact r o l e of t h e i n t e r a c t i o n
between enzymes and t h e p a r t i c u l a t e s t r u c t u r e s of muscle.
* Presented
a t t h e 27th Annual Reciprocal Meat Conference of t h e American
Meat Science Association, 1974.
3 52
To consider t h a t r e v e r s i b l e b i n d i n g - s o l u b i l i z a t i o n plays a r o l e i n
muscle metabolism, s o l u b i l i z a t i o n of a n enzyme must be demonstrated t o
be brought about by conditions which a r e reasonably p h y s i o l o g i c a l whether
it be a change i n pH, i o n i c s t r e n g t h , o r t h e concentration of a metabolite.
It i s worthwhile t o note t h a t a l l of t h e enzymes which w i l l be discussed
can be s p e c i f i c a l l y s o l u b i l i z e d by e i t h e r a s u b s t r a t e , product, o r an
e f f e c t o r of t h e p a r t i c u l a r r e a c t i o n sequence i n which t h e enzyme i s
involved.
The f i r s t enzyme which we examined and t h e one with which w e have
done t h e most work i s l a c t a t e dehydrogenase. We chose t h i s enzyme
because e a r l i e r s t u d i e s had i n d i c a t e d t h a t it w a s approximately an
average enzyme i n i t s degree af binding t o t h e membrane of t h e erythrocyte
(Green e t a l . , 1965). Lactate dehydrogenase i s a t e t r a m e t r i c enzyme
composed of two d i f f e r e n t subunits giving f i v e isoenzymic forms which
a r e labeled as isoenzymes 1, 2, 3 , 4 and 5 depending on t h e i r p a r t i c u l a r
subunit makeup. L D H l contains a l l h e a r t - t y p e o r H subunits and l a c t a t e
dehydrogenase 5 contains f o u r M o r muscle-type s u b u n i t s . Lactate dehydrogenase reoxidizes NADH using p p v a t e a s t h e e l e c t r o n acceptor,
producing l a c t a t e and regenerating t h e oxidized form of t h e c o f a c t o r ,
NAD+, f o r u t i l i z a t i o n by glyceraldehyde-3-phosphate dehydrogenase. The
l a t t e r enzyme is v e r y s e n s i t i v e t o i n h i b i t i o n by NADH.
Lactate dehydrogenase of chicken b r e a s t muscle i s m e of t h e e a s i l y
s o l u b i l i z e d enzymes that I r e f e r r e d t o above. It i s very s e n s i t i v e t o
s o l u b i l i z a t i o n by i n c r e a s i n g pH as shown i n Figure 1 where a n i n f l e c t i o n
p o i n t i s observed around 7.2. This i s i n t h e p h y s i o l o g i c a l range. It
i s a l s o very s e n s i t i v e t o s o l u b i l i z a t i o n by i o n i c s t r e n g t h as shown i n
Figure 2 . The i o n i c s t r e n g t h required f o r s o l u b i l i z a t i o n i s much l e s s
t h a n what i s u s u a l l y considered p h y s i o l o g i c a l . I do n o t intend i n t h i s
paper t o g e t i n t o a d e t a i l e d d i s c u s s i o n of hypotheses on t h e l o c a t i o n
and form of t h e ions i n t h e muscle c e l l . S u f f i c e it t o s a y t h a t t h e r e
i s one school of thought that considers most i f not a l l of t h e ions i n
t h e muscle c e l l t o be complexed t o p r o t e i n s (Ling, 1962, 1969; Cope,
1967). It i s perhaps more t o t h e p o i n t t h a t under conditions which
approximately d u p l i c a t e t h o s e in situ, it can be demonstrated t h a t t h e
enzyme i s capable of binding t o t h e u l t r a s t r u c t u r e of t h e muscle c e l l
(Ross, 1973). This work i s i l l u s t r a t e d by d a t a shown i n Table 1. The
l a c t a t e dehydrogenase a c t i v i t y i s expressed on t h e basis of t h e amount
of water i n e i t h e r t h e muscle or t h e p r e s s j u i c e . Chicken b r e a s t muscle
was f i r s t centrifuged t o remove t h e p r e s s j u i c e , and t h e a c t i v i t i e s
obtained a r e given i n t h e t a b l e . The pressed muscle w a s then imbibed
i n a pH 7.5 b u f f e r of i o n i c s t r e n g t h 0.2, which should be more than
enough t o s o l u b i l i z e t h e enzyme according t o our in v i t r o s t u d i e s . A
small amount of t h e enzyme leaches out during t h i s process but i n any
case, t h e a c t i v i t y on a per m l water b a s i s i n t h e imbibed muscle goes
down s i n c e it i s t a k i n g up b u f f e r with no enzyme i n it. The a c t i v i t y
i n t h e imbibed muscle is a l s o lower t h a n t h a t in t h e f i r s t p r e s s j u i c e .
This imbibed muscle now i s soaked f o r various t i n e periods i n t h e
o r i g i n a l p r e s s j u i c e . Assuming t h a t t h e muscle segments a r e f r e e l y
permeable t o t h e l a c t a t e dehydrogenase i n t h e p r e s s j u i c e , an equilibrium
PER CENT LDH IN SUPERNATANT FRACTION
354
0
3
5
sd
In
0
Ln
0
0
5M
c
a,
k
$
I
V
-4
F:
0
.rl
k
0
Lo
c\I
c!
0
0
e
0
0
\c)
k\O
OCh
(
I
0
0
r
I
0
03
I
0
(0
I
0
T
I
0
nl
Ln
0
0
6,
0
355
TABLE 1. REINTRODUCTION OF LDH INTO PriESSED CHICKEN BHEAST MUSCLE
Sample
A c t i v i t y (umoles/min) per ml water i n
Muscle
Press j u i c e
Whole muscle
1745
Pressed muscle
2517
Imbibed muscle
793
la4
Restored muscle
5hr
1570
900
24 hr
1945
587
The phosphate b u f f e r used for imbibition w a s a t pH 7.5 and a n i o n i c
s t r e n g t h of 0.2. The experimentally determined i o n i c s t r e n g t h of t h e
p r e s s j u i c e w a s approximately t h e same a s t h e b u f f e r .
should be reached between t h e muscle and t h e press j u i c e . T'nat is, on
a per m l of water b a s i s , t h e a c t i v i t i e s i n both t h e muscle and t h e
p r e s s j u i c e should be t h e same. This, i n f a c t , does not happen, b u t
t h e muscle t a k e s up much more enzyme t h a n would be p r e d i c t e d based on
an equilibrium d i s t r i b u t i o n . We i n t e r p r e t t h i s excess enzyme taken up
t o be due t o binding of t h e enzyme t o t h e u l t r a s t r u c t u r e of t h e muscle.
This happens even though t h e i o n i c s t r e n g t h and pH were such t h a t t h e
enzyme would have been s o l u b i l i z e d i n v i t r o . Thus, under conditions
similar t o t h o s e encountered i n s i t u , chicken b r e a s t muscle LDH can
apparently bind t o t h e s u b c e l E l a r p a r t i c u l a t e s t r u c t u r e s .
Figure 3 i l l u s t r a t e s t h e observation t h a t l a c t a t e dehydrogenase
or more LDH5,
from chicken b r e a s t muscle, which i s approximately 9%
i s s o l u b i l i z e d by i t s c o f a c t w , NADH. It i s also important t o n o t i c e
t h a t t h e e f f e c t i v e n e s s of NADH as a s o l u b i l i z i n g agent i s h i g h l y dependent
on t h e a m u n t of t h e i n s o l u b l e muscle p r o t e i n which i s p r e s e n t i n t h e
suspension. That i s , i t s e f f e c t i v e n e s s decreases as t h e amount of t h i s
insoluble protein increases.
S e v e r a l o t h e r metabolites have been examined f o r t h e i r a b i l i t y t o
s o l u b i z e IDH, b u t none of them were as e f f e c t i v e as shown in Table 2
with t h e exception of NADPH (Melnick and H u l t i n , 1970). The oxidized
form of t h e cofactor, NAD+, was a l e s s e f f i c i e n t s o l u b i l i z i n g agent of
t h e enzyme.
100
80
n
u
m
60
5"Io
I
n
-
-I
1" l o
40
0
0
20
10"10
0
0
I
002
I
0.04
!
0.06
NADH, mM
2O"/O
I
0.0
8
I
0.1
Fig. 3
S o l u b i l i z a t i o n of LDH f r m t h e p a r t i c u l a t e f r a c t i o n of homogenized chicken b r e a s t muscle
a s a f u n c t i o n of NADH concentration a t various l e v e l s of t i s s u e p a r t i c u l a t e s i n 10 mM imidazole
b u f f e r a t pH 7.0. The t i s s u e concentrations a r e equivalent t o t h o s e corresponding t o (w/v>
homogenates
A 1% preparation contains approximately 2 mg of i n s o l u b l e p r o t e i n per ml.
.
357
TABLE 2 .
SOLUBILIZATION OF D H BY CELL METABOLITES
STRUCTURALLY REXATED TO NADH
~~
Met ab0 lit el
$ LDH i n supernatant
None
5 -5
NADH
87 *7
NAD+
39 94
NADPH
90 -7
NADP+
32.2
ATP
14.9
ATP + Mg+2
23.2
Adenosine
6.2
Nicotinamide
6.8
Pyrophosphate
22.8
The concentration of a l l metabolites used was 0.4 mM.
6.7, obtained with 10 mM imidazole b u f f e r .
The pH w a s
Lactate dehydrogenase 3, which i s t h e hybrid form with 2 M subunits
and 2 H subunits binds t o t h e p a r t i c u l a t e s t r u c t u r e s of muscle. It i s
s o l u b i l i z e d by t h e same f a c t o r s a s s o l u b i l i z e l a c t a t e dehydrogenase 5 ,
t h a t i s pH, i o n i c strength, and NADH. It i s , however, s o l u b i l i z e d a t
lower pH's, lower i o n i c s t r e n g t h s , and i s p a r t i c u l a r l y s u s c e p t i b l e t o
s o l u b i l i z a t i o n by NADH (Nitisewojo, 1972).
Lactate dehydrogenase 1, which i s composed of t h e f o u r H subunits
does not bind to t h e p a r t i c u l a t e f r a c t i o n o f e i t h e r s k e l e t a l muscle or
h e a r t muscle under any of t h e conditions that we have examined so f a r .
It i s assumed t h a t t h i s i s due t o t h e f a c t t h a t t h e charge d i s t r i b u t i o n
on t h e enzyme i s s i m i l a r t o that on t h e p a r t i c u l a t e s t r u c t u r e s s o t h a t
t h e y a r e not oppositely charged and do not i n t e r a c t . It is probable
t h a t t h e muscle-type subunit i s responsible f o r t h e i n t e r a c t i o n of t h e
enzyme with t h e c e l l p a r t i c u l a t e s t r u c t u r e s .
U p t o this point, we had worked on t h e hypothesis that "soluble"
enzymes l i k e LDH were permanently associated w i t h t h e u l t r a s t r u c t u r e
of the c e l l . %sed on t h e binding data, it became more reasonable t o
expect t h a t t h e r e w a s a r e v e r s i b l e b i n d i n g - s o l u b i l i z a t i o n between t h e
3 53
enzyme and t h e p a r t i c u l a t e s t r u c t u r e s which could be c o n t r o l l e d by
environmental f a c t o r s . The next s t e p , t h e r e f o r e , was t o determine w h a t
k i n e t i c d i f f e r e n c e s t h e r e were between t h e enzyme when bound and when
i n t h e s o l u b l e phase and t o determine i f t h e r e was any l o g i c a l reasons
f o r d i f f e r e n c e s t h a t might be observed.
To do t h i s , we s t u d i e d chicken b r e a s t muscle l a c t a t e dehydrogenase
bound and s o l u b l e . We used a stopped-flow procedure (Ehmann and H u l t i n ,
1973a) so t h a t we could keep t h e enzyme concentration reasonably high t o
d u p l i c a t e t h e p h y s i o l o g i c a l conditions. We also employed a s e n s i t i v e
end-on-photomultiplier tube so t h a t we could use f a i r l y h i g h l e v e l s of
i n s o l u b l e s t r u c t u r a l p r o t e i n which, a s shown above, was necessary t o
m a i n t a i n t h e enzyme i n t h e bound s t a t e i n the presence of t h e c o f a c t o r ,
NADH. We c a r r i e d out t h e s e s t u d i e s over a range of temperatures from
40 t o '
4 C t o r e p r e s e n t roughly t h e range from cold s t o r a g e of t h e
muscle t o t h a t of t h e p h y s i o l o g i c a l temperature.
I n Fig. 4 a r e shown t h e Arrhenius p l o t s of s o l u b l e and bound
l a c t a t e dehydrogenase a t equal enzyme c o n c e n t r a t i o n s . The e f f e c t of
temperature on t h e enzyme i n both phases was similar, but t h e bound
enzyme a t equal concentrations was l e s s a c t i v e by a f a c t o r of approximately 3 or 4 than t h e soluble enzyme.
F i g , 5 shows a p l o t of KM versus temperature f o r bound and s o l u b l e
IDH. There was a small b u t c o n s i s t e n t i n c r e a s e of
f o r t h e bound
enzyme a t a l l temperatures compared t o t h e s o l u b l e . This i n d i c a t e s
t h a t t h e s o l u b l e enzyme has s l i g h t l y more a f f i n i t y for s u b s t r a t e t h a n
does t h e bound. There i s a s i g n i f i c a n t change i n % of both bound and
s o l u b l e IJDH w i t h temperature.
I n h i b i t i o n of LDH can occur through t h e formation of a n a b o r t i v e
t e r n a r y complex of enzyme, NAD' and pyruvate (Zewe and F r o m , 1962),
and t h e formation of t h i s a b o r t i v e t e r n a r y complex has been suggested
as a c o n t r o l mechanism f o r t h e r e g u l a t i o n of IDH a c t i v i t y i n s i t u
(Kaplan e t al., 1968). We s t u d i e d t h e e f f e c t of binding on t h e
formation of t h e a b o r t i v e t e r n a r y complex by a stopped-flow procedure
i n which t h e NAD+ was incubated with t h e enzyme p r i o r t o t h e assay.
We did t h i s s i n c e i n t h e c e l l t h e r a t i o of NADf t o NADH i s h i g h (Long,
1961) ; t h u s , pre -incubat ion should be more phys i o l o g i c a l t h a n NAD+
generation during t h e r e a c t i o n . We adjusted t h e incubation t i m e of t h e
enzyme w i t h NAD' such that maximal i n h i b i t i o n was a t t a i n e d . The time
required v a r i e d as a f u n c t i o n of temperature, t a k i n g longer f o r t h e
r e a c t i o n t o be completed a t t h e lower temperatures (Ehmann and Hultin,
1973b). The results of t h e s e s t u d i e s a r e shown i n F i g . 6, which p l o t s
t h e percentage a c t i v i t y of t h e enzyme i n t h e presence of NAD' t o t h a t
i n i t s absence versus pyruvate concentration. Again we d i d t h i s a t
temperatures of 4, 16, 23 and 4OOC, u s i n g chicken b r e a s t muscle LDH,
that is, LDH5. The amount of i n h i b i t i o n with t h e s o l u b l e enzyme w a s
much g r e a t e r t h a n t h a t of t h e bound enzyme and t h e r e w a s l i t t l e e f f e c t
of temperature. I n t h e case of bound LDH5, however, t h e r e was a
d i f f e r e n c e depending on t h e temperature. A t 40°, which i s c l o s e t o
--
3 59
r
n
/
_I
w
_I
m
3
J
/
P
x
3.00
2.50
BOUND LDH
2.00
1.50
LDH
1.00
0.50
0
I
5
I
10
I
15
I
20
I
25
TEMPERATURE "C
I
30
I
35
I
40
Fig. 5 . Relationship between t h e I(M of s o l u b l e and bound chicken breast muscle LDH and
temperature.
I
0
0
0
T
I
I
I
1
1
I
1
I
I
I
I
I
I
I
I
I
I
I
t
0
0
0
7
I
I
I
aco a8 d 00
0
+CIVN-OA
+CIVN-'A
I
O
0
OtO
(
t h e p h y s i o l o g i c a l temperature of t h e chicken, t h e r e was e s s e n t i a l l y no
i n h i b i t i o n . There was a s i g n i f i c a n t amount of i n h i b i t i o n a t t h e lower
temperatures, b u t it was much l e s s than w a s observed w i t h t h e soluble
enzyme. It i s apparent that t h e binding of I;DH from chicken b r e a s t muscle
t o t h e p a r t i c u l a t e f r a c t i o n does give it some p r o t e c t i o n f r m i n h i b i t i o n
by pyruvate and NAD'.
We c a r r i e d out similar s t u d i e s on t h e p r o t e c t i o n of l a c t a t e dehydrogenase 3 from i n h i b i t i o n by pyruvate and NAD' on binding. There w a s no
evidence of any p r o t e c t i o n i n t h e case of t h i s isozyme (Nitisewojo, 1972).
Obiously, t h e presence of t h e two h e a r t - t y p e subunits i n LDH3 h a s a
profound e f f e c t on t h e a b i l i t y of t h e enzyme t o i n t e r a c t w i t h NAD+ and
pyruvate.
Fig. 7 gives a summary of o u r hypothesis f o r t h e c o n t r o l mechanism
of l a c t a t e dehydrogenase based on t h e k i n e t i c and binding data which we
have obtained t o d a t e ( H u l t i n 5 g.,1972). Glycogen or glucose i s t h e
energy source o f muscle v i a g l y c o l y s i s . Oxidat ion of glyceraldehyde -3phosphate t o 1,3 -diphosphoglycerate by glyceraldehyde -3 -phosphate
dehydrogenase r e q u i r e s NAD+ and produces NADH. The l a t t e r must be
remved s i n c e it is a very p o t e n t i n h i b i t o r of glyceraldehyde-phosphate
dehydrogenase. There i s competition between t h e e l e c t r o n t r a n s p o r t
chain of t h e mitochondria and LDH f o r t h e e l e c t r o n s of NADH. The
former i s more e f f i c i e n t and should be t h e pathway of choice under most
c o n d i t i o n s . It i s t h a t way o r d i n a r i l y because of low NADH l e v e l s i n
r e s t i n g muscle. L;DH i s bound and r e l a t i v e l y i n a c t i v e . When oxygen i s
depleted, as i n s t r e s s , o r i n postmortem muscle, mitochondrial oxidations
cease. The 'NADH l e v e l i n c r e a s e s , s o l u b i l i z e s t h e LDH which can then
r e o x i d i z e t h e NADH more e f f i c i e n t l y t h a n t h e bound LDH
This mechanism
allows t h e maintenance of g l y c o l y s i s under anerobic conditions while a t
t h e same time minimizing competition f o r e l e c t r o n s between LDH and
mitochondria under aerobic c o n d i t i o n s . The d a t a on i n h i b i t i o n by NAD'
and pyruvate would i n d i c a t e t h a t although t h e s o l u b l e enzyme i s potent i a l l y more a c t i v e , it i s a l s o more s u b j e c t t o c o n t r o l . On t h e o t h e r
hand, t h e bound enzyme, although i n h e r e n t l y l e s s a c t i v e , can function
even i n t h e presence of h i g h l e v e l s of NAD' as would be expected t o be
p r e s e n t in r e s t i n g muscle.
.
F i g . 8 i n d i c a t e s a n added f a c t o r , which i s t h a t under any p a r t i c u l a r
s e t of conditions of s u b s t r a t e concentration, pH, e t c . , a whole range
of a c t i v i t i e s of LDH i s p o s s i b l e depending on t h e amount of enzyme t h a t
i s bound as is i l l u s t r a t e d i n t h i s f i g u r e by t h e arrow. Thus t h e r e l a t i v e
a c t i v i t y of l a c t a t e dehydrogenase could be c o n t r o l l e d by f a c t o r s o t h e r
than t h e metabolites t a k i n g part in t h e r e a c t i o n . An example of t h i s
would be a change i n t h e i o n i c environment of t h e c e l l i n t h e a r e a where
t h e enzyme i s f u n c t i o n h g .
W
e examined next t h e glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) enzyme from chicken b r e a s t muscle. We chose t h i s one f o r
s e v e r a l reasons. F i r s t of a l l it has many s i m i l a r i t i e s t o LDH. Besides
being a dehydrogenase, it i s composed of four subunits and has approximately t h e same molecular weight. I n a d d i t i o n , it is f u n c t i o n a l l y
LACTATE
M ITOCHONDRIA
OXIDATION
f
f
f
H + PYRUVATE
4
TRIOSE
PH0S P HAT E S
3-PGADH
\ f
f
f
193-DPG
Fig. 7 . Proposed scheme f o r the r o l e of r c v e r s i b l e b i n d i n g - s o l u b i l i z a t i o n of lactate
dehydrogenase i n carbohydrate metabolism.
2.71
/
0.9-
0-8
0.7
0-6
0.5-
0.40.3 -
O-*I
0.1
1-00 1 2 3 4 5 6 7 8 9 1 0
I
I
I
I
I
[PYRUVATEIX lO4M
-f
30
. 8. I n i t i a l v e l o c i t y of l a c t a t e dehydrogenase as a function of pyruvate concentration i n t h e
o l u b l e ( 0 ) and bound ( 0 ) phase. The arrm r e p r e s e n t s t h e p o t e n t i a l range of a c t i v i t y of t h e
nzyme a t t h a t p a r t i c u l a r pyruvate concentration dependent on the percentage of enzyme t h a t i s
n t h e bound phase.
r e l a t e d t o 1acta.te dehydrogenas ; t h e two work i n c l o s e rapport with
LDH serving t o regenerate cofac )r f o r glyceraldehyde -3 -phosphate
dehydrogenase. Like D E , glyce: Jdehyde-3-phosphate dehydrogenase was
shown t o be a "loosely" bound e r q m e of t h e muscle p a r t i c u l a t e f r a c t i o n
(Dagher, 1971). It was s o l u b i l i z e d somewhat more e a s i l y t h a n t h e LDH
The pH s o l u b i l i z a t i o n curve was very similar t o t h a t o f LDH except t h e
i n f l e c t i o n p o i n t of approximately 7.0 was j u s t s l i g h t l y lower than t h a t
of LDH. NAD+ did not s o l u b i l i z e t h e enzyme a t any l e v e l s t u d i e d while
t h e reduced coenzyme, NADH, only worked a t a very high non-physiological
concentrations. On t h e o t h e r hand, both t h e s u b s t r a t e , glyceraldehyde3-phosphate, and 2,3-diphosphoglycerate had s p e c i f i c s o l u b i l i z i n g e f f e c t s
on t h e enzyme.
.
We again examined some k i n e t i c c h a r a c t e r i s t i c s of t h e enzyme when
bound and when i n t h e s o l u b l e phase. Results of i n i t i a l v e l o c i t y vs
( S ) a r e shown i n F i g . 9 . The soluble enzyme shows s u b s t r a t e i n h i b i t i o n
while t h e bound does n o t . A summary of some of t h e k i n e t i c constants
o f GAPDH i s shown i n Table 3 . The V1dm value i s decreased on irmaobil i z a t i o n a s for LDH but t h e KM value i s decreased a s w e l l . The
i n h i b i t o r constants show t h a t s o l u b l e enzyme i s considerably more
s u b j e c t t o i n h i b i t i o n by NADH than t h e bound. We a l s o have evidence
t h a t a p o r t i o n of t h e glyceraldehyde-3-phosphate dehydrogenase of
chicken b r e a s t muscle i s bound i n s i t u (Melnick and H u l t i n , 1973).
.
We suggest Ynat modification of t h e k i n e t i c p r o p e r t i e s of GAPDH by
s o l u b i l i z a t i o n or binding may be important i n t h e r e g u l a t o r y mechanisms
of t h e muscle c e l l . The lower KM value of t h e bound enzyme f o r glyceraldehyde-3-phosphate compared t o t h e s o l u b l e form enables t h e bound
enzyme t o operate c l o s e t o i t s maximal performance a t low s u b s t r a t e
concentrations. In r e s t i n g muscle, low g l y c o l y t i c a c t i v i t y p r e v a i l s
b u t in working muscle t h e r e i s an i n c r e a s e i n t h e g l y c o l y t i c rate.
This i n c r e a s e i n t h e r a t e of g l y c o l y s i s leads t o higher concentrations
of many o f t h e g l y c o l y t i c intermediates including glyceraldehyde-3phosphate. If t h e s e compounds are involved in s o l u b i l i z a t i o n Df
glyceraldehyde-3-phosphate dehydrogenase & s i t u , t h e enzyme would be
s o l u b i l i z e d . The enzyme i n s o l u b l e form has a higher m a x i m a l v e l o c i t y
and seems b e t t e r s u i t e d t o function i n such a s i t u a t i o n , t h a t i s , t h a t
of high s u b s t r a t e concentration. The soluble enzyme i s a l s o more
s e n s i t i v e t o i n h i b i t i o n by NADH; thus t h e c e l l would have b e t t e r
c o n t r o l of t h e a c t i v i t y of t h i s enzyme under conditions where i t s
maximal a c t i v i t y is high.
Arnold and P e t t e (1970)have shown t h a t f r u c t o s e diphosphate,
dihydroxyacetone phosphate, ATP, ADP and P i i n h i b i t t h e absorption of
a l d o l a s e t o a c t i n within t h e physiological range of concentrations of
t h e s e components. They have also shown t h a t binding of a l d o l a s e t o
for f r u c t o s e diphosphate
F - a c t i n doubles the V m and increases t h e
by almost one order of magnitude. Aldolase appears t o be an e a s i l y
s o l u b i l i z e d enzyme, being completely removed by moderate concentrations
of s a l t .
366
-+
.-
3c
W
0
5 2c
>
.-
I
0.2
0
0
I
0.4
A
0.
I
0.6
[ G 3 d mM
I
0.8
.o
1
1
Fig. 9 . Initial velocities of soluble znd bound glyceraldehyde3-phosphate dehydrogenase as a function of glyceraldehyde-3phosphate concentration. The concentration of enzyme was 5 uM
in all cases (Dagher and Hultin, 1973).
367
TABLE 3. KINETIC PARAMETERS OF SOLUBLE AND BOUND
GLYCERALDEHYDE -3 -PHOSPHATE DMYDROGENASE
Soluble
v-,
nmoles/sec
29
Bound
7
The concentration of enzyme w a s 5 uM i n all c a s e s .
Hexokinase and phosphofructokinase r e p r e s e n t t h e c l a s s of enzymes
r e q u i r i n g more vigorous conditions t o s o l u b i l i z e than t h e ones previously
mentioned. Hexokinase i s t h e r a r e g l y c o l y t i c enzyme t h a t has been
considered t o e x i s t a t l e a s t p a r t l y bound t o s u b c e l l u l a r s t r u c t u r e s .
This has been due t o two f a c t o r s , one i s t h e aforementioned f a c t that
it i s not e a s i l y s o l u b i l i z e d , and second, it i s a s s o c i a t e d with mitochondria, and mitochondria have been s t u d i e d from many t i s s u e s i n some
depth.
For t h i s reason, hexokinase, i n both t h e soluble and bound form,
has been s t u d i e d t o q u i t e some e x t e n t . The k i n e t i c parameters o f Q
f o r glucose and ATP, and a l s o V m comparisons between bound and
s o l u b l e enzymes, have v a r i e d a g r e a t d e a l depending on t h e source of
t h e enzyme. It has been demonstrated r e p e a t e d l y however, t h a t t h e
s o l u b l e enzyme i s much more s e n s i t i v e t o i n h i b i t i o n by glucose-6phosphate t h a n i s t h e bound enzyme.
To s o l u b i l i z e hexokinase, r e l a t i v e l y vigorous conditions a r e
r e q u i r e d . Combinations of reasonably d r a s t i c pH's, high s a l t concent r a t i o n s , ( - 0 . 8 ~ ) and d e t e r g e n t s ( o f t e n with s o n i c a t i o n ) , w i l l give
good s o l u b i l i z a t i o n (Southard and H u l t i n , 1972a). Hexokinase can a l s o
be s o l u b i l i z e d by low l e v e l s of
osphate and considerably
higher l e v e l s of ATP (Rose and
t h e l a t t e r being a s u b s t r a t e
of t h e r e a c t i o n and t h e former a product.
We found about 6@ of t h e hexokinase of chicken b r e a s t muscle t o
be a s s o c i a t e d with the. p a r t i c u l a t e f r a c t i o n . To study t h e e f f e c t of
s o l u b i l i z a t i o n , however, we p u r i f i e d mitochondria and worked s t r i c t l y
with t h e mitochondrial enzyme (Southard and H u l t i n , 1972b). We showed
t h a t t h e mitochondrial enzyme w a s e s s e n t i a l l y a l l one isoenzymic form,
t h e so-called type 11. We s o l u b i l i z e d mitochondrial hexokinase by using
potassium c h l o r i d e a t a f i n a l concentration of 0.8M and c i t r a t e t o a
f i n a l concentration of 10 mM with pH adjusted t o 5.5. We avoided t h e
use of d e t e r g e n t s s h c e t h e s e sometimes have s t r a n g e e f f e c t s on some
enzymes. This suspension then was t r e a t e d with u l t r a s o n i c energy and
then separated by c e n t r i f u g a t i o n . The procedure was repeated which
gave s o l u b i l i z a t i o n of g r e a t e r than 9 6 of t h e t o t a l bound hexokinase.
After s e p a r a t i o n of t h e s o l u b l e enzyme, t h e supernatant f r a c t i o n must
be quickly adjusted t o pH 7 i n order t o maintain a c t i v i t y .
Atkinson (1968)has proposed t h e concept of energy charge which i s
defined i n equation (1)and i s r e l a t e d t o t h e amount of high energy
phosphate bond compared t o t h e t o t a l n u c l e o t i d e .
(ATP)
f
1/2 (ADP)
Energy charge = (ATP) + (ADP)
f
(AMP)
Eq* (1)
He has found t h a t many c o n t r o l enzymes a r e s e n s i t i v e t o t h i s f a c t o r .
The response of t h e enzyme i s u s u a l l y such t h a t t h e a c t i v i t y of an enzyme
involved with energy generation decreases w i t h increased energy charge
and t h e a c t i v i t y of one involved with energy u t i l i z a t i o n increases with
increased energy charge. I n t h e l i v i n g muscle c e l l , t h e energy charge
v a r i e s over t h e range from about 0.8 t o 0.9, although t h i s w i l l drop
postmortem. The response of bound and s o l u b i l i z e d mitochondrial hexokinase from chicken b r e a s t muscle t o energy charge which we have determilled
as shown
i n Fig. 10. Over t h e p h y s i o l o g i c a l range, bound hexokinase
i s much more s e n s i t i v e than i s t h e s o l u b l e although t h e d i f f e r e n c e
decreases a t t h e low energy charges which one would see with time
postmortem. We have shown that t h i s d i f f e r e n c e i n response t o energy
charge i s a r e f l e c t i o n of a s i g n i f i c a n t l y g r e a t e r i n h i b i t i o n of t h e
bound enzyme by ADP than t h e s o l u b l e (Southard and H u l t i n , 1972b).
The g e n e r a l shape of t h e hexokinase response curve t o energy
charge i n d i c a t e s that it t a k e s p a r t p r i n c i p a l l y i n an e n e r g y - u t i l i z i n g
pathway. This means t h a t t h e r e a c t i o n catalyzed by t h e enzyme c o n t r i b u t e s t o a b i o s y n t h e t i c pathway, t h a t i s , one r e q u i r i n g a n e t expenditure
of energy. Thus, t h e glucose-6-phosphate produced by t h e hexokinasecatalyzed r e a c t i o n i n muscle should be p r i n c i p a l l y u t i l i z e d for glycogen
s y n t h e s i s r a t h e r t h a n g l y c o l y s i s . When glycogen i s synthesized,
s i g n i f i c a n t l e v e l s of glucose-6-phosphate must be p r e s e n t s i n c e t h i s
compound a c t i v a t e s t h e glycogen s y n t h e s i s system. A s discussed above,
t h e s o l u b l e enzyme i s more s e n s i t i v e t o glucose-6-phosphate i n h i b i t i o n
than i s t h e bound. Therefore, t h e bound enzyme wauld be b e t t e r s u i t e d
f o r producing glucose -6 -phosphate for glycogen synthes is than t h e
s o l u b l e . The a s s o c i a t i o n of hexokinase with t h e mitochondrion may
occur i n o r d e r t o e f f i c i e n t l y supply ATP t o t h e enzyme f o r glycogen
s y n t h e s i s . A r e c e n t study has shown, i n f a c t , t h a t mitochondrial
hexokinase can u t i l i z e ATP produced by mitochondria more e f f i c i e n t l y
than exogenously added ATP (Got6 e t a1
1972).
--.,
When glycogen s t o r e s a r e complete, t h e r e i s no need t o manufacture
more. If t h e energy charge were high, t h e enzyme would be a c t i v e and
waste ATP. It would a l s o produce glucose-6-phosphate, but t h i s i s a
r e l a t i v e l y poor i n h i b i t o r of bound hexokinase. A p o s s i b l e explanation
f o r t h e c o n t r o l of hexokinase under t h e s e conditions would be t h e
IO0
80
60
40
,a
I
I
0.2
0.4
0.6
0.8
Energy Charge
F i g . 10. E f f e c t of energy charge m hexokinase a c t i v i t y of t h e
mitochondrial-bound and s o l u b i l i z e d enzvme .
I.o
370
s e n s i t i v i t y of bound hexokinase t o energy charge. A s ATP i s u t i l i z e d
and t h e energy charge drops, t h e bound enzyme i s i n h i b i t e d . T h i s would
provide t h e c o n t r o l t o maintain energy s t o r e s without a t t h e same time
wasting energy. This suggests t h a t perhaps s o l u b l e hexokinase t a k e s
p a r t p r i n c i p a l l y i n g l y c o l y s i s and i s regulated by glucose-6 -phosphate
while t h e bound enzyme may be involved i n glycogen s y n t h e s i s and i s
c o n t r o l l e d by t h e energy charge. The p a r t i c u l a r l o c a t i o n of t h i s enzyme
i n t h e c e l l would a f f e c t not only i t s k i n e t i c p r o p e r t i e s b u t a l s o t h e
metabolic sequence i n which it i s a c t i v e . This could have profound
s i g n i f i c a n c e f o r t h e r a t e of g l y c o l y t i c a c t i v i t y i n muscle postmortem.
Many consider phosphofructokinase t o be t h e prime p o i n t of
r e g u l a t i o n of g l y c o l y t i c a c t i v i t y i n muscle. We have found t h a t
e s s e n t i a l l y a l l of t h e PFK from chicken b r e a s t muscle i s i s o l a t e d with
t h e sedimentible f r a c t i o n a f t e r homogenization (Chism, 1973). Like
hexokinase, it r e q u i r e s rather d r a s t i c treatments t o s o l u b i l i z e i t .
It i s extremely s e n s i t i v e t o i n a c t i v a t i o n by many handling procedures
and, t h u s , t h e evaluation of i t s r o l e i n soluble and bound forms i s
extremely d i f f i c u l t . High phosphate helps t o m a i n t a i n s t a b i l i t y . The
pH-solubility graph of PIX i s shDwn i n F i g . 11. It i s almost a
completely cooperative response. Phosphofructokinase w a s a l s o
s o l u b i l i z e d by one of i t s s u b s t r a t e s and e f f e c t o r s , ATP. The concent r a t i o n dependence of t h i s i s shown i n Fig. 12. This s o l u b i l i z a t i o n
by ATP i s time-dependent w i t h a h a l f -time of approximately 20-30 s e c
Phosphofructokinase i s also s o l u b i l i z e d i n a time-dependent manner by
ADP, b u t t h i s t a k e s w e l l over h a l f an hour f o r 5& s o l u b i l i z a t i o n , and
we do not f e e l t h a t t h i s i s a p h y s i o l o g i c a l l y s i g n i f i c a n t phenomenon.
.
Kinetic s t u d i e s of phosphofructokinase i n t h e bound and s o l u b l e
and V m could be c a l c u l a t e d ,
form i n any d e t a i l such that values of
have not been p o s s i b l e because of t h e d i f f i c u l t i e s of d e a l i n g with t h i s
enzyme. However, we have shown that t h e bound form i s more a c t i v e t h a n
t h e s o l u b l e . We have a l s o shown t h a t t h i s enzyme can rebind t o t h e
p a r t i c u l a t e f r a c t i o n . The conditions which lead t o s o l u b i l i z a t i o n ,
i .e., h i g h pH and high (ATP), a r e t h o s e which would be found in t h e
r e s t i n g c e l l . The enzyme would be s o l u b l e and l e s s a c t i v e . On
d e p l e t i o n of ATP, and dropping of t h e pH, such as would occur p o s t mortem o r under o t h e r types of s t r e s s such as e x e r c i s e , t h e enzyme
would be rebound and would be more a c t i v e and presumably would i n c r e a s e
t h e r a t e of g l y c o l y t i c a c t i v i t y under t h e s e conditions r e l a t i v e t o t h e
s o l u b l e form of t h e enzyme. Again, our proposal is that r e v e r s i b l e
b i n d i n g - s o l u b i l i z a t i o n r e s u l t s i n c o n t r o l of t h e a c t i v i t y of t h e enzyme.
I n s u m m a y , we s e e sane b a s i c s i m i l a r i t i e s i n many g l y c o l y t i c
enzymes (and probably o t h e r s o l u b l e enzymes as w e l l ) regarding t h e
e f f e c t of r e v e r s i b l e i n t e r a c t i o n s with c e l l u l a r m r t i c u l a t e s t r u c t u r e s .
These a r e :
1. The s u b s t r a t e s , products, o r e f f e c t o r s of t h e r e a c t i o n s a r e
capable of s o l u b i l i z i n g the enzymes.
2. There i s a r e v e r s i b l e b i n d i n g - s o l u b i l i z a t i o n under conditions
which a r e reasonably p h y s i o l o g i c a l .
PH
11. S o l u b i l i z a t i o n of phosphofructokinase from t h e
p a r t i c u l a t e f r a c t i o n of homDgenized chicken b r e a s t muscle
as a function of pH.
372
100
80
13
Lll
N
0
r/)
40
0
\
O
20
0
0
.
.
1
I
I
2
I
3
I
4
I
I
5
mM ATP
Fig. 12. Solubilization of phosphofructokinase from the particulate
fraction of homogenized chicken breast muscle as a function of ATP
concentration.
3 73
3 . There a r e very o f t e n d i f f e r e n t p r o p e r t i e s i n t h e unbound and
bound forms. I should note h e r e , however, t h a t these types of
d i f f e r e n c e s a r e not mandatory f o r t h e r e t o be a n e f f e c t i v e n e s s o f
binding. For example, i f l a c t a t e dehydrogenase and glyceraldehyde-3phosphate dehydrogenase were bound t o t h e same s u r f a c e , j u s t t h e very
f a c t o f t h e i r being c l o s e l y aligned and a b l e t o pass s u b s t r a t e from
one t o t h e o t h e r quickly and e a s i l y , might be s u f f i c i e n t j u s t i f i c a t i o n
f o r t h e i r binding without any modification of t h e p r o p e r t i e s of t h e
i n d i v i d u a l enzymes.
4. The enzymes involved a r e subunit enzymes which, a t l e a s t under
some conditions, a r e a l l o s t e r i c c o n t r o l enzymes, and
5 . There i s d i f f i c u l t y i n studying t h e s e e f f e c t s under reasonably
-i n vivo
conditions, and very o f t e n under non i n vivo conditions t h e s e
d i f f e r e n c e s i n p r o p e r t i e s are not observed. I a m s u r e t h a t t h i s i s one
reason why more i n s t a n c e s of t n e s e occurrences have not been r e p o r t e d .
Along w i t h t h e s e b a s i c s i m i l a r i t i e s , t h e r e may a l s o be important
d i f f e r e n c e s between enzymes. There may be d i f f e r e n t s p e c i f i c changes
i n t h e k i n e t i c parameters of i n d i v i d u a l enzymes. It i s a l s o l i k e l y
t h a t one w i l l see d i f f e r e n c e s depending on t h e p a r t i c u l a r s u r f a c e t o
which t h e enzyme b i n d s . There i s no reason t o assume, f o r example,
t h a t t h e same p r o p e r t i e s are going t o be observed when LDH binds t o
mitochondria as when it binds t o t h e nucleus o r t o a c t i n .
The r a t e s of s o l u b i l i z a t i o n may a l s o vary. We have done some work
which i n d i c a t e s t h a t t h e s o l u b i l i z a t i o n o f LDH i s extremely f a s t , f a s t e r
t h a n w e can measure with a stopped-flow apparatus s o t h a t it must be a t
l e a s t i n t h e milli-second range. On t h e o t h e r hand, both hexokinase and
phosphofructokinase have a r e l a t i v e l y slow s o l u b i l i z a t i o n r a t e with
half-times of t h e order of half a minute. The r a t e of s o l u b i l i z a t i o n
o r binding may i t s e l f play a r o l e in t h e c o n t r o l of t h e enzyme ( G u m a a
and McLean, 1969).
Another important consideration i n t h e e f f e c t s of t h e changes i n
enzyme c h a r a c t e r i s t i c s on binding in meat i s t h e s p e c i f i c e f f e c t t h a t
temperature has. With l a c t a t e dehydrogenase, t h e e f f e c t of temperature
i s extremely complex. The d i f f e r e n c e isoenzymes behave d i f f e r e n t l y ,
and the bound and s o l u b l e enzymes respond i n d i f f e r e n t ways t o changes
i n temperatures (Ehmann and H u l t i n , 1973a,b,c; Nitisewojo, 1972).
I have presented in t h i s paper some d a t a Dbtained i n our l a b o r a t o r y
concerning i n t e r a c t i o n s of various g l y c o l y t i c enzymes and c e l l u l a r
p a r t i c u l a t e s t r u c t u r e s as w e l l as some of our working hypotheses as t o
t h e r a t i o n a l e f o r t h e s e i n t e r a c t i o n s . The suggestions have involved t h e
f u n c t i o n i n g of l i v i n g muscle s i n c e it i s u n l i k e l y that t h e g l y c o l y t i c
enzymes p a r t i c i p a t e i n a programed d e s t r u c t i o n o f t h e muscle c e l l a f t e r
death. It i s my b e l i e f , however, t h a t a f u l l understanding of t h e
f u n c t i o n i n g of g l y c o l y s i s i n muscle postmortem, and t h e changes dependent
on g l y c o l y s i s , r e q u i r e s a knowledge of t h e e f f e c t s of t h e i n t e r a c t i o n
of t h e enzymes and t h e p a r t i c u l a t e s t r u c t u r e s of t h e c e l l .
374
This work w a s supported by funds from t h e U.S. Public Health
Service (Grant GM-l2064), t h e National Science Foundation (Grant GB
21262), and t h e Massachusetts Experiment S t a t i o n .
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M. L, Greaser: What r m t e r i a l a r e you using t o bind your enzyme?
You r e f e r t o it as p a r t i c u l a t e m a t e r i a l .
H. 0 . H u l t i n : It has v a r i e d considerably depending on t h e
p a r t i c u l a r enzyme. The b e s t example i s l a c t i c dehydrogenase, which w e
have bound t o a whole t o t a l c e l l homogenate, t o a c t i n , and t o carboxymethylcellulose. You do g e t d i f f e r e n c e s i n t h e k i n e t i c p r o p e r t i e s of
t h e enzyme depending on t h e nature of t h e m a t e r i a l s t o which it binds,
and w e f e e l t h i s i s due p r i n c i p a l l y t o t h e charge c h a r a c t e r i s t i c s of
t h e binding s u r f a c e .
M . L . Greaser: So you've been using mainly muscle homogenates and
t h e p a r t i c u l a t e m a t e r i a l a f t e r you've washed out s u b s t r a t e s and so on?
H . 0. Hultin: \!e t a k e out very l i g h t f r a c t i o n s and v e r y heavy
f r a c t i o n s because o f mechanical problems through t h e stop-flow. I t i s
a mixture, containing most a l l of t h e components.
M. L . Greaser: Is it your idea, then, that t h e s u b c e l l u l a r
f r a c t i o n s t h a t most people prepare by conventional methods contain a
l a r g e amount of t h e g l y c o l y t i c enzymes?
H. 0 . H u l t i n : We have done some work on t h i s . AB an example, we
can prepare mitochondria which by t h e u s u a l c r i t e r i a seem t o be i n
p r e t t y good condition. If we feed them any of t h e phosphorylated sugars
l i k e glucose -6 -phosphate, f r u ct os e -6 -phosphate o r f r u c t o s e diphosphate,
t h e y perform as w e l l i n r e s p i r a t i o n a s with s u c c i n a t e , so t h e g l y c o l y t i c
enzymes a r e not t h e l i m i t i n g f a c t o r i n t h e mitochondria. The exception
i s hexokinase, which i s much slower i f given glucose.
C . E . Allen: Could you give us a l i t t l e more s p e c i f i c information
on LDH-V i n r e l a t i o n t o temperature postmortem, where we g e t i n t o q u a l i t y
problems?
H. 0 . Hultin: This i s v e r y d i f f i c u l t t o summarize, and t h e b e s t
answer I can give i s t h a t i t ' s u n p r e d i c t a b l e . For one of t h e iso-enzymes,
LDH-1, t h e r e i s no d i f f e r e n c e i n a c t i v i t y between 23 and 40°, f o r LDH-111
it i s intermediate, and f o r LEH-V t h e d i f f e r e n c e i s very l a r g e . The
e f f e c t i v e d i f f e r e n c e between LDH-1U. and LDH-V, which f o r t h e s o l u b l e
forms i s f a i r l y s i g n i f i c a n t , tends t o disappear on binding, because
binding d o e s n ' t a f f e c t LDH-111 very much but a f f e c t s LDE-V very d r a s t i c a l l y ,
.
C E . A l l e n : Would you c a r e t o r e l a t e t h e temperature-dependent
accumulation of calcium t o postmortem cooling r a t e ?
H . 0 . Hultin: The c l a s s i c a l explanation of t h e cold-shortening
e f f e c t i s based on two f e a t u r e s , one t h e a b i l i t y of t h e sarcoplasmic
reticulum f r a c t i o n t o maintain calcium i n t h e bound s t a t e , t h e o t h e r
the c o n t r a c t i l e apparatus i t s e l f . If t h e s e two have d i f f e r e n t temperature
dependencies, t h e r e a r e going t o be some temperatures a t which one of
t h e reac8ions d m i n a t e s over t h e o t h e r , s o t h a t a s you lower t h e temperature,
say t o 5 C, ;you can g e t a r e v e r s a l i n t h e r e l a t i v e importance of t h e two
systems. I would j u s t l i k e t o throw i n one coment, an idea which i s
not n e c e s s a r i l y r e l a t e d t o t h i s question: we have seen with a f a i r
number of enzymes t h a t t h e r e i s sometimes a very s i g n i f i c a n t drop i n
t h e K, of t h e enzyme with temperature. This means t h a t i f t h e concent r a t i o n of s u b s t r a t e i n t h e muscle i s of t h e order of magnitude of t h e
$, which it most l i k e l y i s , t h e n as you lower t h e temperature you may
not be lowering t h e a c t i v i t y of t h e enzyme. We see t h i s a l i t t l e b i t
w i t h chicken b r e a s t muscle LDH, which has e x a c t l y t h e same a c t i v i t y a t
400 a s a t 4' provided you use l o w pyruvate concentration. Now with high
pyruvate concentration, you s e e a n Arrhenius-type r e l a t i o n s h i p , w i t h
about a seven-fold d i f f e r e n c e . But if you lower t h e s u b s t r a t e concent r a t i o n down t o w h a t i s most l i k e l y i n t h e muscle, t h i s d i f f e r e n c e
disappears. It i s amazing J u s t how l i t t l e work has been done on
temperature e f f e c t s on enzymes.
.
B B. Marsh: Thank you very much, Herb, f o r a s t i m u l a t i n g paper
and a n a c t i v e d i s c u s s i o n .
* * *
379
H . F . Bernholdt: I want t o welcome you t o the Food Service
Committee's session t h i s afternoon and I want t o thank i n p a r t i c u l a r
t h e C a m i t t e e members t h a t worked with us through t h e year t h a t helped
put t h i s program t o g e t h e r on t h e Challenges and Opportunities i n t h e
Fmd Service Industry. I am s u r e we a l l recognize t h e impwtance of
t h e Food Service i n d u s t r y t o t h e meat i n d u s t r y a n d t o t h e food i n d u s t r y
i n g e n e r a l . With our mobile population, with t h e unique problems of
feeding people on t h e move or i n p a r t i c u l a r s i t u a t i o n s such a s h o s p i t a l s
o r i n s t i t u t i m s , it i s a challenging a r e a - - i t ' s a f a s t mcnring a r e a . I n
f a c t , t h i s afternoon it i s s o fast-moving t h a t one of our speakers
cancelled. The f i r s t speaker 3 n t h e program was t o be from t h e a i r l i n e
i n d u s t r y and unfortunately due t o o t h e r p r i o r i t i e s they had t o c a n c e l .
However, I f e e l t h a t i n s p i t e of t h a t we have a well-rounded and f u l l
program t h a t c e r t a i n l y w i l l be of i n t e r e s t t o a l l of us h e r e . It i s
unfortunate t h a t t h e a i r l i n e peoFle a r e not represented because t h e i r s
i s a unique i n d u s t r y and t h e i r problems a r e unique. C e r t a i n l y t h e
l o g i s t i c s of feeding i n an a i r l i n e s i t u a t i o n present many, many problems.
I would recommend t o f u t u r e Food Service Committees t h a t t h e y consider
agair, including t h i s t o p i c of a i r l i n e feeding i n t h e i r program. I would
also suggest s i n c e t h e y have expressed a n i n t e r e s t t h a t one r,f t h e s e
speakers be from S b Chefs i n New York C i t y who said that given a l i t t l e
time and a l i t t l e opportunity they would be very, very happy t o p a r t i c i p a t e i n t h e American Meat Science Association meeting. So I have very
quickly dispatched of t h e a i r l i n e i n d u s t r y and we w i l l m m e on t 3 our
second t o p i c - - t h a t of t h e i n s t i t u t i o n a l t r a d e . We have with us today
M r Bob A l s t r i n of Armour and Company. Bob i s a graduate of Cornell
U n i v e r s i t y with a B.S. from t h e Hotel Restaurant Administretion School
and, of course, knows many of our f r i e n d s t h e r e including our good and
departed f r i e n d , J e r r y Wanderstock. He was i n t h e Navy a s a p i l o t from
1956 t o 1959. From 1959 t r , 1968, Bob w a s General Manager of s e v e r a l
S t a u f f e r Restaurants i n Chicago and Cleveland. He was a l s o Director of
Restaurant Operations on a venture team with General M i l l s from 1968 t o
1970. He i s p r e s e n t l y S a l e s Manager f o r Prepared Foods and Portion
Control Meats f o r Armour and Company located i n t h e i r Phoenix g e n e r a l
o f f i c e . I n a d d i t i o n , he served on t h e Board of Directors of t h e Chicago
I l l i n o i s Restaurant Association from 1967-1968. So we have with us a
man who has long time experience i n t h e food s e r v i c e i n d u s t r y and w i l l
t a l k t o us today on " I n s t i t u t i o n a l Feeding."
.