Download A comparative study of glycolysis in red and white muscles of the

Reprinted from J. Fish Bioi. (1977) 11, 575-588
A comparative study of glycolysis in red and white muscles
of the trout ( Salmo gairdneri)
and mirror carp
( Cyprinus
carpio )
IAN A. JOHNSTON*
Research Unit for Comparative
University
of Bristol,
Bristol
Animal
BS8 lUG,
Respiration,
Avon, England
(Accepted 2 September 1976)
The activities of some glycolytic and associated enzymes have been determined in the
muscles of trout and carp to investigate the possibility that the discrepancies previously
reported between lactate accumulation and anoxic tolerance in these two fish result from
underlying differences in glycolytic potential. Steady state concentrations of certain
glycolytic intermediates were also determined in freeze-(;lamped muscles from tankrested fish. The activities of hexokinase, phosphorylase and phosphofructokinase were
approximately 2-3 times lower in carp than trout white muscles. pyruvate kinase and
lactate dehydrogenase activities were 5 times lower in carp white muscle. The lower,
broader pH optima of lactate dehydrogenaseand pyruvate kinase from carp compared to
trout muscles is thought to be correlated with the greater anoxic tolerance of the carp.
Glycolytic enzyme profiles were markedly different between the red and white muscles
of the rainbow trout but broadly similar, with the exception of hexokinase activity, for
the corresponding muscles of the carp. The results are discussed in relation to what is
known about anaerobiosis in these two species and the comparative physiology of red
and white muscles in fish.
I. INTRODUCTION
The bulk of the myotomal muscle mass in teleosts is composed of fast twitch fibres
which are poorly supplied with mitochondria and blood capillaries (Bilinski, 1975).
In common with most vertebrate tissues, the white skeletal muscle offish responds to
periods of anoxia by an increase in anaerobic glycogenolysis. However, there is
considerable variation in the ability of different fish species to withstand hypoxic
conditions and this is often correlated with their ecology (Marvin & Heath, 1968;
Hughes, 1973). For example, trout live in environments of high oxygen content and
only have a limited ability to survive hypoxic exposure. On the other hand various
carp species, which are adapted to spending periods at extremely low oxygen tensions,
have the capacity for extended anaerobiosis (Blazka, 1958). Interestingly, the
accumulation of lactate in the white muscles of these two species does not appear to
be correlated with the lengths of time each can sustain anoxia. Rainbow trout exposed
to an environmental oxygen level of 40 torr for 1 h accumulated 15.6llmollactate!g
white muscle. In contrast, Crucian carp maintained under similar conditions for
1.5 h accumulated only 3 llmollactate!g muscle (Johnston, 19750, b). This difference
in the accumulation of lactic acid is not accounted for by differences in the transport
of anaerobic endproducts through the general circulation (Johnston, unpublished
.Present addressand addressfor correspondence:
Departmentof Physiology,Universityof St.
Andrews,St. Andrews,Fife, Scotland.
575
..
576
I.
A. JOHNSTON
results). Similar discrepancies between lactate accumulation and anaerobic work
load have been reported for another carp species, Cyprinus carpio (Driedzic &
Hochachka, 1975).
'.
Dreidzic & Hochachka (1975) have explored the possibility that the low rate of
lactate accumulation in the carp white muscle during hypoxia could be explained by
the existence of alternative anaerobic pathways to glycolysis in this tissue as occurs in
the muscles of numerous facultative anaerobes and in diving mammals (Magnum &
van Winkle, 1973; Hochachka, Owen, Allen & Whit tow, 1975). In these animals,
during anaerobiosis, carbohydrates and amino acids are utili sed simultaneously with
the production of a variety of endproducts (Hochachka, 1975). The coupling of two
mitochondrial energy yielding reactions to glycolysis allows both an increase in the
high energy phosphate equivalents and the maintenance of redox balance within the
cell (Hochacllka, Fields & Mustafa, 1973; Hochachka, 1975). However, no evidence
has been found for the accumulation of multiple anaerobic endproducts in the white
muscle of either the mirror (Driedzic & Hochachka, 1975) or crucian carp (Johnston,
1975a).
In the present study the activities of some glycolytic and associated enzymes have
been determined in the muscles of the trout and mirror carp to investigate the possibility that there is an enzymic basis which might account for the discrepencies in
endproduct accumulation between these two species.
n. MATERIAlS AND MEmoDS
FISH
Mirror carp (Cyprinus carpio L.) about 20 cm long were obtained during the spring from a
commercial fishery. They were acclimatised at 14° C for several weeks in tanks of filtered,
circulated fresh water. During this period they were fed regularly on a diet of chopped pigs
heart. Rainbow trout (Salmo gairdneri Richardson) of similar length were obtained from
another fish farm during the same period. The trout were also acclimatised at 14° C before
being used for analyses. Trout were regularly fed on a commercial brand offish pellets. No
food was given to either species in the 24 hour period prior to their being sacrificed.
DISSECTION OF MUSCLES
Fish were lightly anaesthetizedin a 0.1 g/1solution of MS 222 (Sandoz Ltd) and killed by
stunning. Red muscle was dissectedfrom both sides of the body in the region of the lateral
line. Care was taken to dissect only the most superficial layers of red muscle to avoid contamination with other fibre types (Johnston, Patterson, Ward & Goldspink, 1974). It is
particularly important to exclude pink fibres from the dissection as these have beenshown to
have very different metabolic characteristics from the red fibres (Johnston, Davison &
Goldspink, 1976). White fibres were dissectedfrom the deep epaxial muscle adjacent to the
dorsal fin.
DETERMINATION OF GLYCOLYTIC INTERMEDIATES
Small pieces of muscle (total weight 0.5-1 g) were immediately freeze clamped in liquid
nitrogen. The frozen tissue was pulverized in a stainless steel pestle and mortar cooled in
liquid nitrogen and extracted at 0-40 C in 0.6 N perchlorate. Tissue debris was removed by
centrifugation and an aliquot of the clear supernatant was neutralised with KOH and made
1 mM with respectto Tris-HCI pH 7-1. Sampleswere stored at -35° C until analyses.
Glucose 6-phosphate was assayedby a spectrophotometric method (Barrett & Beis, 1973)
in 1 ml of 50 mM glyclyglycine buffer pH 7.6 containing 150 jlmol MgSO" 5 jlmol NADP ,
0.2 jlmol glucose-6-phosphatedehydrogenaseand 0.2 mI of neutral perchlorate extract. The
increase in extinction of 340 nm wavelength was measured on completion of the reaction.
Glucose-1-phosphateand fructose-6-phosphate were determined in the same cuvette by
.
G L YCOL
YSIS
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MUSCLES
577
addition of phosphoglucomutase
(2 ~g) and glucose-phosphate isomerase respectively
(Barrett & Beis, 1973). Lactate and pyruvate were determined enzymatically by the methods
of Hohorst (1965) and Landon, Fawcett & Wynn (1962) respectively.
PREPARATION
OF HOMOGENATES
Approximately
0.5 9 samples of freshly excised muscle were cut into small pieces and
homogenised at 0.4° C in an MSE top-drive homogeniser for 5 periods of 20 s with cooling.
The following media were used for individual enzymes:
Phosphorylase (EC 2.4.1.1) 3 vols of 100 mM maleate-NaOH
buffer, 1 mg/ml bovine
serum albumin.
Hexokinase (EC 2.7.1.1) 3-5 vols of 50 mM Tris-HCI,
5 mM EDTA,
2 mM MgCI.,
1 mM dithiothreitol
pH 7.5.
Phosphofructokinase
(EC 2.7.1.11) 9 vols of 15 mM Tris-HCI,
1 mM EDTA, SmM
MgSO. pH 7.5.
pyruvate kinase (EC 2.7.1.40) 10 vols of25 mM Imidazole-HCI pH 7.0.
Lactate dehydrogenase (EC 1.1.1.27) 20vols of 100mM sodium phosphate buffer
pH7.6.
Homogenates were centrifuged for 12 000 g for 20 min at 4° C and the supernatants retained
for enzyme assay.
ASSA Y OF ENZYME
ACTIVITY
Preliminary experiments were carried out to determine the optimal conditions of substrates, co-ions and pH for each of the enzymes under study. To minimise degradative
changes occurring in the muscle homogenates, assays of enzyme activity were carried out
within 15 min of preparation of the homogenates. The assay conditions for the individual
enzymes were as follows.
Phosphorylase. Phosphorylase was assayed according to the method of Hedrick & Fischer
(1965). Aliquots of supernatant were incubated at 20° Cat a final concentration of 100 mM
Maleate-NaOH
buffer pH 6.8 (trout), pH 6.6 (carp), 75 mM glucose-l-phosphate,
1%
glycogen, 1 mM AMP, 5 mM dithioerythritol,
0.5 mg/m1 bovine serum albumin. Blanks
were performed in which AMP was omitted from the reaction mixture. The reaction was
terminated with 6% (w/v) HCIO, after 1-5 min under conditions in which the release of Pi
was linear with respect to time. Liberated phosphate was assayed by the method of AIlen
(1940).
Hexokinase. Hexokinase was assayed spectrophotometrically utilising an A TP regenerating
system and monitoring the change in extinction of NADH at 340 Dm wavelength with
respect to time. The reaction medium contained 85 mM Tris-HCI pH 7.7, 8 mM MgCI"
0.8 mM EDTA,
1 mM glucose, 0.4 mM NADP+,
10 mM phosphorylcreatine,
100 ~g
creatine phosphokinase and 100 ~g of glucose-6-phosphate dehydrogenase. Control assays
contained the above medium with glucose omitted.
Phosphofructokinase.
Phosphofructokinase
was assayed spectrophotometrically
in a
medium of 50 mM Tris-HCI pH 7-7 (trout) or pH 7-6 (carp), 1.5 mM Fructose-6-phosphate,
1.5 mM ATP, 25 mM KCI, 2 mM MgCI" 0.15 mM NADH and excess aldolase, triose
phosphate isomerase, and glycerophosphate dehydrogenase.
Pyruvate kinase. pyruvate kinase was assayed by measuring the decrease in extinction of
NADH with respect to time at a wavelength of 340 nm in a medium of 100 mM imidazoleHCI pH 7-4 (trout) or pH 6.5 (carp), 70mM KCI, 4 mM MgCI" 2mM ADP, 1.5 mM
phosphoenol pyruvate, 0.16 mM NADH and excess lactate dehydrogenase.
Lactate dehydrogenase. Lactate dehydrogenase was assayed spectrophotometrically
in a
medium of 35 mM phosphate buffer pH 7-5, 1.6 mM sodium pyruvate and 0.27 mM NADH.
Lactate dehydrogenase isoenzyme ratio was defined as the ratio between the two activities
in the presence of low (0.33 mM) and high (10 mM) pyruvate concentrations (Wilson,
Cahn & Kaplan, 1963).
AIl measurements of enzyme activity were performed at 20° C in duplicate with appropriate
enzyme and rejgent blanks. Results were expressed as ~mol substrate utilised, g fat free
dry weight of muscle -1, min -1. Similar enzyme activityratios between muscles were obtained
when the results were expressed as umoles. me suDernatant orotein- -1 min -1- Protein
GL YCOL YSIS
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579
MUSCLES
TABLE I. Statistical
analyses of the ratio of activities of some glycolytic and associated
enzymes in the white and red muscles of the rainbow trout and mirror carp
Enzyme
Rainbow trout
ratio activities
white/red
mu~cles
Phosphorylase
Hexokinase
Phosphofructokinase
pyruvate kinase
Lactate dehydrogenase
2.8
0.47
2.5
2.9
4.9
NS signifies not significantly
Prob.
sig. diff.
Mirror carp
ratio activities
white/red
muscles
Prob.
sig. diff.
P<O.Ol
P<O.OOl
P<O.OOl
P<O.OOl
P<O.OOl
0.92
0.17
1.1
1.2
1.1
NS
P<O.O5
NS
NS
NS
different at the P=O.O5 level of probability,
concentrations were determined using a biuret method (Gornall, Bardawill & David, 1949).
Fat free dry weights of muscles were determined as previously described (Johnston &
Goldspink, 19730).
STATISTICAL ANALYSES
The data was tested for significance using an analysesof variance method.
All the biochemical reagents and enzymesused in this study were obtained from the Sigma Chemical
Co. Ltd. (London).
m. RESUL TS
The activities of some glycolytic and associated enzymes under saturating conditions
of substrates and co-ions and at optimal pH for red and white myotomal muscles of
trout and carp are given in Figs land 2. Considerable species differences occurred
in the relative activities of these enzymes both between red and white muscles and
between homologous muscles.
Hexokinase activities were higher in the red than white myotomal muscles of both
species. However, the activity of the trout muscle enzyme was three times higher than
that from the carp (Figs 1 and 2). The effect of pH on hexokinase activity was similar
for both species with a maximal activity occurring at around pH 7.7.
TABLE II.
Statistical analyses of the ratio of activities of some glycolytic and associated
enzymes in homologous muscles of the rainbow trout and mirror carp
Enzyme
Red muscle
ratio activities
trout/carp
Phosphorylase
Hexokinase
0.87
Phosphofructokinase
pYruvate kinase
Lactate dehydrogenase
0.93
1.22
1.85
1.17
NS signifies-not significantly
Prob.
sig. cliff.
White muscle
ratio activities
trout/carp
Prob.
sig. cliff.
NS
NS
NS
P<O.OOl
NS
2.67
3.36
2.22
4.65
5.2R
P<O.Ol
P<O.OOl
P<O.OOl
P<O.OOl
P<O.OOl
different at the P=O'OS level of probability.
~RO
I.
TABLE III.
A.
JOHNSTON
Lactate dehydrogenase activities and isoenzyme ratios in the red and white
muscles of the rainbow trout and mirror carp
Mean:!: S.E. enzyme activity (~moles, g-l, min-I)
Rainbow trout
'Mirror carp
Red muscle
White muscle
Red muscle
White muscle
Assay
cnndit.inn"
0.33 mM pyruvate
10 mM pyruvate
Isoenzyme ratio
3800::1:390
3140::1:295
1.'1
996::!:132
532::!:64
1.87
722:!:82
409:!:59
1.76
657:!:113
485:!:54
1.1~
Phosphorylase and phosphofructokinase activities were approximately 2-3 times
lower in the carp white muscle than the trout white muscle. The ratio of activities of
these two enzymes between white and red muscles was about 2-3 for the trout. However, the activities of these enzymes were broadly similar in both the red and white
muscles of the mirror carp (Tables I and II). Phosphorylase showed a sharp pH
profile in the muscles of both these species. Maximal activity was observed at pH
6.8 for the red and white muscles of the trout and at pH 6.4 and pH 6.6 respectively
for the red and white muscles of the carp. Phosphofructokinase activity showed a
bell shaped pH curve with maximal activities in the range pH 7.7-7.8 for the trout and
pH 7.6 for the carp.
pyruvate kinase and lactate dehydrogenase (LDH) activities were 4.5-5 times lower
in carp than trout white muscle. Pyruvate kinase activities were also significantly
lower in carp red muscle (Table II). Lower lactate dehydrogenase isoenzyme activities
were found in the white than red myotomal muscle for both species(P<O.O5 ; Table III).
However, the activities of this enzyme were not significantly different between the
red and white muscles of the mirror carp (Table II; Fig. 2). The LDH isoenzyme
ratios of homologous muscles were not found to be significantly different between
species (P=O.O5 level). There were major differences between species in the effects of
pH on enzyme activity for these two enzymes. Trout muscle pyruvate kinase had a
pronounced optima at pH 7.4 [(Fig. 3(a)]. In contrast, the activity of the carp muscle
'a)
Trout
It'\~~
an('
"\-0
/
..p
SOC'
/
.,
0-0
/
'.
.-.
./
/
1,1'
0"1
.,
-
~
I
---
S
7
A
pH
FIG. 3. The effect of pH on maximal enzyme velocity (Vo) for pyruvate kinase from the red (.) and
white (0) muscles of rainbow trout and mirror carp. Assay conditions are given in the text.
-
GL YCOL YSIS
! ( a)
IN
FISH
MUSCLES
581
Trout
4000
d
p-
~
200<'
/
I
7
~
8
pH
FIG. 4. The effect of pH on maximal enzyme velocity ( V0) for lactate dehydrogenase from the red (.)
and white (0) muscles of the rainbow trout and mirror carp. As~ay conditions are given in
the text.
v
GLY
G6P
GIP
F6P
PYR
LAC
FIG. S. Steady state concentrations
of certain glycolytic intermediates in the red (dark stippled
columns) and white (light stippled columns) muscles of rainbow trout (upper histogram) and
mirror carp (lower histogram).
Concentrations
are given as ~moles, g dry weight muscle-1,
g -1. The number of fish analysed is given above the columns.
.
582
I.
A.
JOHNSTON
enzyme was fairly independent of pA in the range pA 6.2-7.5 with an optima at
pA 6.5 (Fig. 3(b)). The pA optima of LDA from the trout muscles was in the range
pA 7.3-7.7 (Fig. 4(a)). A rather broader pA profile was obtained for the corresponding
enzyme from the carp with near optimal activity in the range ,pA 6.9-7.8.
The steady state contents of certain glycolytic intermediates in freeze clamped
muscles from trout and carp are given in Fig. 5. Glucose-6-phosphate concentrations
were higher in the red than white muscles of both fish species (P<O.O5). Concentrations of this intermediate were higher in the corresponding trout than carp muscles
(P<O.00l). Glucose-l-phosphate concentrations were 8-15 times lower than those
for G6P and .there were no significant differences found between species or muscles.
Fructose-6-phosphate levels were approximately 3 times higher in trout than carp
white muscle (P<O.Ol). Pyruvate concentrations were 2-3 times higher in white than
red muscle for both species (P<O.Ol). Lactate concentrations were only higher in the
white than red muscles in the case of the trout (P<O.OI). In resting fish lactate
concentrations were 2-3 times higher in the trout than carp white muscle (P<O.OOI).
IV. DISCUSSION
The Jateral musculature of fish consists of a complex arrangement of twitch muscles.
Unlike most other vertebrates the different fibre types in fish are usually arranged in
anatomically discrete regions. Slow red fibres are a minor component and form a
triangular block of muscle adjacent to the lateral line. These fibres have a predominantly oxidative type of metabolism being abundantly supplied with myoglobin,
capillaries, mitochondria and the associated enzyme systems (Bilinski, 1975). The
remaining bulk of the muscle consists mostly of large diameter fast twitch fibres
which have relatively few mitochondria and wbich rely on anaerobic metabolism for
their energy supply (Love, 1970; Bilinski, 1975). A third type of fibre only occurs in
very small numbers in the rainbow trout (Johnston, Davison & Goldspink, 1975a)
but constitutes around 10% of the muscle bulk in the mirror carp (Davison, Goldspink
& Johnston, 1976). These so-called pink fibres have an intermediate oxidative
capacity between the red and the white fibres and are believed to be of the fast twitch
type (Johnston et al., 1976).
The activity of an enzyme under different physiological conditions in viva will
depend on such factors as substrate availability, the concentration of allosteric
modulators and local H+ concentrations at the active site. Studies of enzyme activity
measured under optimal conditions in vitra reveal nothing about the activity of the
enzyme under different physiological states but do give an estimate of maximum
glycolytic potential. Measurements of phosphofructokinase activity, a rate limiting
enzyme of glycolysis, have been used to give an estimate of maximum glycolytic flux
in vertebrate muscles (Crabtree & Newsholme, 1972; Newsholme & Start, 1973).
In the present study the in vitra activities of certain glycolytic enzymes have been
determined for trout and carp muscles to investigate the possibility that the discrepancies observed between lactate accumulation during hypoxia and the anoxic
tolerance of these species (Johnston, 1975a, b; Driedzic & Hochachka, 1975) are
attributable to differences in the glycolytic potentials of the muscles.
Hexokinase is unique among glycolytic enzymes in that its activity in vertebrate
muscles is directly related to the pigmentation and respiratory capacity of the muscles
(Burleigh & Schimke, 1969). Thus the hexokinase activities of vertebrate twitch muscles
h"vp hppn ~hnwn tn he 2-10 times hi2her in red than white muscles (Burleigh &
GL YCOL YSIS
IN
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MUSCLES
583
Schimke, 1969; Crabtree & Newsholme, 1972). Similar findings have been obtained
for both fish species in the present study. The red muscle in rainbow trout and mirror
carp is thought to be primarily concerned with providing the muscular effort associated
with slow speed swimming (Hudson, 1973; Johnston et a/., 1976). The higher
activities of hexokinase in fish red muscle may reflect the preferential utilisation of
glucose from the liver rather than muscle glycogen for energy production during
this type of locomotory activity. In vitro activities of hexokinase have therefore been
used to estimate the maximum rate of glucose utilisation during low speed swimming
(Crabtree & Newsholme, 1972). Such estimates have been found to approximate to
theoretical rates of carbohydrate utilisation derived from measurements of oxygen
consumption of the whole animal (Newsholme & Start, 1973). Evidence from experimental studies on swimming fish, however, suggest the oxidation of lipids in the red
muscle supplies a major part of the energy requirements during low speed swimming
(Bilinski, 1975). Hexokinase is also unique among glycolytic enzymes in that it
shows large exercise induced changes in activity in both red and white muscles (Peter ,
Jeffress & Lamb, 1968). Hexokinase activity increased to 200 % in various mammalian
muscles subjected to endurance training or different activity levels (Baldwin, Winder ,
Terjung & Holloszy, 1973). Differences in hexokinase activity presented in the present
study between trout and carp white muscles (Table II) may therefore partly represent
differences in activity patterns during captivity rather than true interspecific variation.
However, it is clear that in general the glycolytic enzyme profiles of trout and carp
red and white muscles differ considerably (Table I). Glycogen stores and steady state
concentrations of certain glycolytic intermediates in red and white muscles were also
different between species (Fig. 5). The higher activities of glycolytic enzymes in trout
white than red muscles is characteristic of the metabolic differentiation of fast and
slow twitch fibres in vertebrate striated muscles (Newsholme & Start, 1973). In
contrast, glycolytic enzymes were found to have similar activities in both the red and
white muscles of the mirror carp (Fig. 2). Similar results have been obtained for the
red and white muscles of the yellow eel, Angui//a angui//a (Bostrom & Johansson,
1972). The yellow eel like the carp is a sluggish, bottom dwelling animal. However,
the active sexually differentiated migratory stage of the eel was found to have three
times the activity of glycolytic enzymes in the white as red muscle (Bostrom &
Johansson, 1972). Although white muscle with its poor blood supply, low mitochondrial content and LDH isoenzyme ratio (Table III) is obviously more adapted for
anaerobiosis than red muscle, the present study indicates that the stereotyped view
of these muscles types as having high and low glycolytic potentials respectively is not
always valid, since, in the case of the carp in the present study the glycolytic potentials
of the red and white muscles would appear to be broadly similar. It is known that a
correlation exists between the degree of red muscle development in the myotome and
sustained swimming performance (Boddeke, Slijper & van der Stelt, 1959). It would
also appear the basic metabolic differentiation of red and white muscles in fish is
subject to evolutionary modification associated with a particular mode of life.
Modifications in the metabolism and division of labour of fish swimming muscles are
also likely to occur with development (Nag & Nurshall, 1972), migration (Bostrom &
Johansson, 1972) and starvation (Johnston & Goldspink, 1973a; Patterson &
Goldspink, 1973; Patterson et a/., 1974).
The higher activities of phosphofructokinase found in the white muscles of trout
relative to carp in the present study probably reflects a greater potential maximum
-
584
I.
A.
JOHNSTON
glycolytic flux in this species (Figs I, 2; Table II). This is correlated with activities of-;
lactate dehydrogenase which were 5 times higher in the trout white muscle. Lactate
dehydrogenase is important in providing a continuous supply of oxidised NAD+
during anaerobic glycolysis, thus maintaining redox balance. Sprints of activity in
fish are produced by the contraction of the vast bulk of white muscle and this quickly
results in fatigue (Bainbridge, 1960; Bone, 1966). Short bursts of swimming in
salmonids results in the mobilisation of 50% of white muscle glycogen stores and the
production of large amounts of lactic acid (Black, Bosomworth & Docherty, 1966).
As with the results of experimentally induced hypoxia there are considerable species
differences in the accumulation of lactate by the white muscles of trout and carp
exercised to fatigue. Lactate increases of 12 ~moles/g have been reported for carp
exercised to fatigue whereas under comparable conditions lactate levels reach in
excess of 50 ~moles/g in trout white muscle (Black, Robertson, Conner, Lam & Chiu,
1962; Stevens & Black, 1966; Driedzic & Hochachka, 1975). The relatively low
glycolytic potential of carp muscle is surprising since it has been calculated that in a
related carp species approaching 80% of the propulsive force is derived anaerobically
during high speed cruising (Smit, Amelink-Koutstaal, Vijvetberg & van Vaupel-Klein,
1972). At these speeds in carp it is likely that contraction of the white fibres provides
the major contribution to locomotion (Johnston & Goldspink, 1973b, c; Johnston,
et al., 1976; Davison, et al., 1976). Furthermore it has been reported the wild carp can
survive several months at near zero Po2 in ice-locked lakes. Under these conditions
the fish do not become torpid due to the low temperature but show thermal compensation involving an increased metabolic rate and increased activities of the enzymes of
energy metabolism and muscle contraction (Hazel & Prosser, 1974; Johnston,
et al., 1975). The lower pH optima of phosphorylase and pyruvate kinase
(Fig. 3) and the higher relative activity of lactate dehydrogenase at low pH in the carp
(Fig. 4) may well reflect the need for a high activity of these enzymes during naturally
occurring periods of anaerobiosis when increases in lactate concentration may result
in a drop in intracellular pH. The low activity of pyruvate kinase in carp red muscle
is of particular interest. In mammals, although there is evidence that pyruvate kinase
is a non-equilibrium enzyme, there is little support for it being a regulatory enzyme
(Newsholme & Start, 1973). In contrast, the pyruvate kinase from the red muscle of
the Cruciancarp is under tight metabolite control (Johnston, 1975c). It is allosterically
regulated, being inhibited by A TP and alanine and activated by low concentrations of
fructose-l,6-diphosphate.
Fructose-l,6-diphosphate also strongly reverses the inhibitory effect of negative modulators. It is interesting that the skeletal muscle of diving
mammals also has a low pruvate kinase activity compared to terrestrial mammals and
furthermore is under metabolite regulation like the carp muscle enzyme (Storey &
Hochachka, 1974a,b). Both of thesegroups of animals have a well developed anaerobic
capacity and the ability to oscillate rapidly between aerobic and anaerobic metabolism.
The highly developed anaerobic capacity of carp muscles would therefore appear
to be incompatible with their relatively low glycolytic potential and low rate of
lactate accumulation during anoxia. Two hypotheses have been advanced to explain
this apparent discrepancy. The first is that there is something missing in our understanding of anaerobic mechanisms in fish (Hochachka & Somero, 1973; Hochachka,
1975; Driedzic & Hochachka, 1975). Succinate and alanine have been shown to
accumulate in the tissues of numerous faculative anaerobes during anoxia in addition
to or in place of lactic acid (Magnum & van Winkle, 1973). There is also evidence
.-
GL YCOL YSIS
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585
for the operation of similar pathways in the muscles of diving mammals following
deep dives (Hochachka et al.. 1975). It has been postulated that the significance of
amino acid fermentation during anoxia lies in the coupling of two mitochondrial
energy yielding reactions to glycolysis. This allows the substrate level p~osphorylation
of high energy phosphate compounds to proceed in the absence of molecular oxygen
without the cell passing into a highly reduced state (Hochachka, 1975).
Evidence for anaerobic mitochondrial energy production in which succinate and
alanine accumulate in the muscles during anoxia has been obtained for carp red
muscle but not white muscle (Johnston. 1975a; Driedzic & Hochachka. 1975). This
difference in the metabolic response to hypoxia between red and white muscles is
probably related to differences in mitochondrial density. The ratio of mitochondria
in red and white muscles of a related carp species expressed as percentage volume
occupied is 23 : I respectively (Patterson & Goldspink. 1973). In crucian carp
succinate only increased to a tenth of the concentrations of lactate in the red muscle
during anoxia (Johnston, 1975a). It would therefore seem that these pathways are
unlikely to provide a very large contribution to the total capacity of the swimming
musculature to do anaerobic work particularly since red fibres are only a minor
component of the myotome in this species (7 %) (Johnston & Goldspink, 1973c). In
addition. there is also evidence for the operation of similar mechanisms in the red
muscle of rainbow trout (Johnston, 1975b).
There is also evidence that anaerobic metabolism in fish is qualitatively different
from that of mammals (Bilinski, 1975). During anaerobiosis numerous workers have
demonstrated the production of CO2 and NH4 of metabolic origin (Hochachka, 1961;
Ekberg, 1962; Kutty, 1968; Dejours, Armand & Verriest, 1968). Furthermore,
unlike mammals, there is evidence that fish can utilise proteins directly to supply
energy for muscular work even under normal nutritional conditions (Mercy Bai,
1970: Kutty, 1968).
The second hypothesis which has been advanced to explain the low accumulation
of lactate in carp white muscle during hypoxia and exercise involves the noncirculatory
transfer of metabolites between red and white muscles (Wittenberger & Deaciuc,
1965; Wittenberger, 1972; Wittenberger. 1973; Wittenberger, Coprean & Morar
1975). Principally this involves a transfer of lactate from the white to red muscle and
of glucose from red to white muscle. The evidence for this comes from the incubation
of isolated muscle pieces in various media with or without glucose containing hormones known to stimulate glucose transport or pharmacological agents which affect
membrane permeability (Wittenberger et al.. 1975). A major difficulty in accepting
that such a direct transfer of metabolites occurrs in viva is that it requires the postulation of novel transport phenomena. In addition. glucose-6-phosphatase activity is
either very low or absent in skeletal muscle and therefore another reaction would be
necessary to produce free glucose from existing muscle glycogen stores (Wittenberger
& Deaciuc, 1970).
Many species of fish have impressive anaerobic capabilities and some may be said
to be examples of vertebrate facultative anaerobes (Blazka, 1958; Hochachka, 1975).
Certainly. sustained anaerobiosis is important in the lives of many groups of fishes
and the elucidation of the anaerobic mechanisms operative in these animals adapting
them for this mode of life is of great interest to the comparative physiologist.
I am grateful to the Natural Environment ResearchCouncil for a postdoctoral Research
Fellowship award and to Professor G. M. Hughes for the provision of facilities.
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586
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A.
JOHNSTON
~
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