Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel

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Braz J Med Biol Res 34(8) 2001

Effect of exercise on glycolytic enzymes in fish tissuesBrazilian Journal of Medical and Biological Research (2001) 34: 1055-1064ISSN 0100-879X

Influence of exercise on the activityand the distribution between free andbound forms of glycolytic and associatedenzymes in tissues of horse mackerel

1Department of Natural Sciences, Precarpathian University, Ivano-Frankivsk, Ukraine2Institute of Biochemistry and Department of Biology, Carleton University, Ottawa,Ontario, Canada

V.I. Lushchak1,*,T.V. Bagnyukova1,*,

J.M. Storey2 andK.B. Storey2

Abstract

The effects of short-term burst (5 min at 1.8 m/s) swimming and long-term cruiser (60 min at 1.2 m/s) swimming on maximal enzymeactivities and enzyme distribution between free and bound states wereassessed for nine glycolytic and associated enzymes in tissues of horsemackerel, Trachurus mediterraneus ponticus. The effects of exercisewere greatest in white muscle. The activities of phosphofructokinase(PFK), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase),and phosphoglucomutase (PGM) all decreased to 47, 37, 37 and 67%,respectively, during 60-min exercise and all enzymes except phospho-glucoisomerase (PGI) and PGM showed a change in the extent ofbinding to subcellular particulate fractions during exercise. In redmuscle, exercise affected the activities of PGI, FBPase, PFK, andlactate dehydrogenase (LDH) and altered percent binding of only PKand LDH. In liver, exercise increased the PK activity 2.3-fold andreduced PGI 1.7-fold only after 5 min of exercise but altered thepercent binding of seven enzymes. Fewer effects were seen in brain,with changes in the activities of aldolase and PGM and in percentbinding of hexokinase, PFK and PK. Changes in enzyme activities andin binding interactions with subcellular particulate matter appear tosupport the altered demands of tissue energy metabolism duringexercise.

CorrespondenceV.I. Lushchak

Department of Natural Sciences

Precarpathian University

57 Shevchenko str.

Ivano-Frankivsk, 76000

Ukraine

E-mail: [email protected]

*On leave from Karadag Natural

Reserve, National Academy of

Sciences of Ukraine, Kurortne,

Feodosia, Crimea, Ukraine

Received April 17, 2000

Accepted May 15, 2001

Key words· Trachurus mediterraneus· Swimming exercise· Glycolysis· Subcellular enzyme binding

Introduction

Horse mackerel (Trachurus mediterra-neus ponticus) are highly active, pelagic fishthat can swim at intermediate speed for longperiods of time as well as in brief high-speedbursts for shorter periods (1). Intensificationof swimming is powered by creatine phos-phate hydrolysis and by ATP synthesis via

anaerobic glycolysis, primarily catabolizingendogenous glycogen, but also utilizing bloodglucose when swimming is prolonged. Theeffects of burst swimming on intracellularmetabolite levels and pH in fish muscle havebeen well documented and include decreasedpH, ATP, creatine phosphate, and glycogenalong with elevated levels of glucose, lac-tate, inorganic phosphate, ammonium ion,

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V.I. Lushchak et al.

AMP and IMP (1-9). In addition, exerciseincreased both total glycogen phosphorylaseactivity and the percentage of enzyme in theactive, a form for an overall 6-fold increasein the amount of active phosphorylase introut white muscle (8). The activation ofenergy metabolism in fish involves severalregulatory mechanisms acting in concert;allosteric regulation, protein phosphoryla-tion and enzyme binding to subcellular mac-romolecules all contribute to the activationof different enzymes (10). The most contro-versial of these mechanisms is reversibleenzyme binding to subcellular macromol-ecules.

Enzyme binding to cellular macromol-ecules has been reported extensively. Forexample, enzymes of glycogen metabolismbind to glycogen particles, AMP-deaminasebinds to myosin, and various glycolytic en-zymes bind to filamentous actin (11-14).The distribution of enzymes between freeand bound forms may, in turn, be modulatedby changes in metabolite and ion levels (15-17) or by reversible protein phosphorylation(18). The physiological relevance of someinteractions is obvious (e.g., glycogen phos-phorylase or synthetase binding to glyco-gen), but the functional significance of gly-colytic enzyme association with F-actin hasbeen the subject of debate (13,17,19). Theseinteractions can change enzyme functionalproperties. For example, bound lactate dehy-drogenase (LDH) is not inhibited by highconcentrations of pyruvate (20,21), and ismore stable to trypsin hydrolysis than thefree enzyme (22). Catalytic properties canalso be modified by enzyme interaction withstructural elements (21-25).

A redistribution of glycolytic enzymesbetween bound and soluble fractions hasbeen noted in several systems as a responseto stimuli that change glycolytic flux (e.g.,exercise, anoxia, hypoxia) (26-28), includ-ing trout white muscle (12,29). In general, anincrease in enzyme binding correlates withan increase in glycolytic flux and vice versa.

But in our recent studies with a teleost fish,the sea scorpion, subjected to hypoxia wefound that binding of some enzymes in-creased whereas binding of other enzymesdecreased under the same stress conditions(30). Therefore, to determine whether thiswas an isolated instance or a common re-sponse by fish muscle, we undertook a com-parable study with another teleost species.

The present study analyzes the effects ofswimming exercise under two regimens,short-term burst exercise and long-termcruiser exercise, on the maximal activitiesand free versus bound distribution of en-zymes in four tissues (brain, liver, and redand white muscle) of the Black Sea teleostfish, the horse mackerel. The results showthat activities and distribution of enzymesare modified during exercise and that theeffects are dependent on the intensity ofswimming.

Material and Methods

Experimental animals

Horse mackerel (Trachurus mediterra-neus ponticus Aleev) weighing 25-40 g werecaught in October 1995 at Karadag Bay,Black Sea (Crimea, Ukraine) and were heldin tanks provided with flowing sea water ona native dark/light cycle and temperature of16 ± 1oC. Fish were not fed and were usedthe next day.

Biochemical reagents and coupling enzymes

All chemicals were obtained from Boeh-ringer Mannheim (Montreal, PQ, Canada),Sigma Chemical Co. (St. Louis, MO, USA)or Reachim (Russia, USSR).

Fish exercise

Fish were gently netted from the holdingtank and transferred to a specially constructedhydrodynamic tube providing linear laminar

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Effect of exercise on glycolytic enzymes in fish tissues

flow of water and equipped with an electricengine as described earlier (1). Eight fishwere used in one experiment which wasstarted about 6-7 am. Initially, water flowwas held at low speed (0.1-0.3 m/s) for 1 h.After that, four fish were removed as con-trols and the rest were exercised for 60 minat a speed of 1.2 m/s (cruiser exercise) be-fore sampling. In previous work from ourlaboratory it was shown that under this regi-men fish may swim for several hours (1).Other fish were exposed to the same 1-h lowspeed swimming but this was then followedby 5 min at a speed of 1.8 m/s (burst exer-cise); fish in this group were exercised andsampled individually. All fish were killedquickly by trans-spinal dissection and tissuesamples were immediately removed and usedfor the preparation of homogenates; this pro-cess took about 2 min. Whole brain andliver, and portions of red and white musclewere used for analysis. White muscle sampleswere always removed from the same posi-tion in the body, just below the first spinalfin.

Preparation of tissue extracts

Exercised tissues were quickly washed inice-cold 0.9% NaCl, blotted on filter paper,weighed and homogenized in a Potter ho-mogenizer with a glass pestle in 10 volumesof homogenization solution containing 0.25M sucrose, 10 mM dithiothreitol (DTT), 20mM NaF, 1 mM EDTA, and 20 mM imidaz-ole-HCl, pH 7.0, at 20oC; 0.1 mM phenyl-methylsulfonyl fluoride (PMSF, a proteaseinhibitor) was added immediately beforehomogenization (30). The homogenate wascentrifuged for 20 min at 45,000 g at 4oC andthe supernatant was removed and stored onice. Enzyme activity in the supernatant rep-resents the free enzyme fraction. The pelletwas rehomogenized in 5 volumes (relative toinitial tissue weight) of the second homog-enization solution with a high salt content torelease bound enzymes (0.25 M sucrose, 10

mM DTT, 20 mM NaF, 1 mM EDTA, 20mM imidazole-HCl and 500 mM KCl). Aftercentrifugation, the supernatant was removedand the pellet was extracted a second timeand centrifuged and the second supernatantwas combined with the first. Enzyme activ-ity in this fraction represents the bound en-zyme fraction. Enzyme activities were quan-tified in both free and bound fractions andthe percent binding was calculated. Recov-ery of total enzyme activity was checked inall cases by comparing the sum of enzymeactivities in free and bound fractions withtotal enzyme activities measured in tissuehomogenates. Recovery was close to 100%in most cases, ranging from 80 to 120%. Themaximal enzyme activities (units/g wetweight) shown in the tables are those meas-ured in whole homogenates.

Measurement of enzyme activities

Maximal activities were assayed at 25oCusing a KFK-324 spectrophotometer (Lomo,Leningrad, Russia) by monitoring the changein absorbance of NADH or NADPH at 340nm. All reaction mixtures contained 50 mMimidazole-HCl, pH 7.0, in a final volume of1.0 ml including the added extract. Blanksdid not contain the most specific substrate(indicated by an asterisk in the assays be-low). Reactions were started by the additionof the specific substrate but, if no spontane-ous change in absorbance was observed,subsequent assays were started by the addi-tion of homogenate. Optimal assay condi-tions were based on those described earlier(30) and adapted to our experiments. Hex-okinase (HK, EC 2.7.1.1): 10 mM glucose(*), 0.2 mM NADP, 2 mM ATP, 5 mMMgCl2, 0.5 unit (U) glucose-6-phosphate de-hydrogenase (G6PDH), and 50 µl superna-tant. Phosphoglucoisomerase (PGI, EC5.3.1.9): 2.5 mM fructose-6-phosphate (*),0.2 mM NADP, 5 mM MgCl2, 0.5 U G6PDH,and 5-10 µl supernatant. Phosphofructoki-nase (PFK, EC 2.7.1.11): 5 mM fructose-6-

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supernatant. LDH (EC 1.1.1.27): 1 mM pyru-vate (*), 0.15 mM NADH, 1 mM EDTA, and1-50 µl supernatant. G6PDH (EC 1.1.1.49):2.0 mM glucose-6-phosphate (*), 5 mMMgCl2, 0.2 mM NADP, and 20-50 µl super-natant. Phosphoglucomutase (PGM, EC2.7.5.1): 15 mM glucose-1-phosphate (*),0.20 mM NADP, 5.0 mM MgCl2, 0.5 UG6PDH, and 20-50 µl supernatant. Fruc-tose-1,6-bisphosphatase (FBPase, EC3.1.3.11): 0.1 mM fructose-1,6-bisphosphate(*), 0.2 mM NADP, 5 mM MgCl2, 0.5 UG6PDH, 0.5 U PGI, and 30-50 µl superna-tant. One unit of enzyme activity is definedas the amount of enzyme that utilizes 1 µmolof substrate per 1 min at 25oC. All values areexpressed as means ± SEM. Differences be-tween means were determined by analysis ofvariance followed by the two-tailed Dunnetttest. For technical reasons, the number ofanimals was limited (3-4 per set) but, evenso, in many cases we obtained statisticallysignificant differences between experimen-tal groups.

Results

The effect of exercise under both regi-mens on the activities of glycolytic and asso-ciated enzymes is shown in Tables 1-4. Inwhite muscle, exercise resulted in a signifi-cant decrease in the activities of severalenzymes (Table 1). Activities of PFK, PKand FBPase decreased under both exerciseregimens, whereas PGM was reduced by33% only after long-term cruiser swimming.After 5-min burst exercise, PFK, PK andFBPase activities were reduced to 72, 46,and 75% of their corresponding control val-ues, but 60 min of sustained exercise had aneven greater effect, lowering activities to 47,36 and 37% of the controls, respectively.The activities of four enzymes were affectedby exercise in red muscle (Table 2). Theactivities of PGI and FBPase decreased sig-nificantly after 5-min burst exercise fallingto 66 and 42%, respectively, of their control

Table 2. Effect of exercise on the activities (units/g wet weight) of glycolytic andassociated enzymes in red muscle of horse mackerel.

Enzyme Control 5-min burst exercise 60-min cruiser exercise

HK 0.42 ± 0.04 0.48 ± 0.03 0.39 ± 0.02PGI 9.45 ± 0.93 6.23 ± 0.92a 9.35 ± 0.36b

PFK 0.17 ± 0.02 1.93 ± 0.61a 0.20 ± 0.01b

Ald 14.2 ± 1.2 11.4 ± 1.9 11.6 ± 1.6PK 136 ± 13 122 ± 4 111 ± 4LDH 31.7 ± 3.3 56.8 ± 5.6a 36.7 ± 1.9b

PGM 1.12 ± 0.04 1.12 ± 0.07 0.93 ± 0.05FBPase 0.55 ± 0.12 0.23 ± 0.08a 0.18 ± 0.01a

Data are reported as means ± SEM of 3-4 determinations in tissue from separateindividuals. aP<0.05 compared to the corresponding control value and bP<0.05 com-pared to the 5-min burst exercise group (two-tailed Dunnett test).For abbreviations, see legend to Table 1.

phosphate (*), 5 mM ATP, 0.15 mM NADH,10 mM MgCl2, 50 mM KCl, 0.5 U aldolase(Ald), 0.5 U triosephosphate isomerase, 2 Uglycerol-3-phosphate dehydrogenase, and 30-50 µl supernatant. Ald (EC 4.1.2.13): 0.5mM fructose-1,6-bisphosphate (*), 0.15 mMNADH, 0.5 U triosephosphate isomerase, 2U glycerol-3-phosphate dehydrogenase, and2-20 µl supernatant. Pyruvate kinase (PK,EC 2.7.1.40): 10 mM phosphoenolpyruvate(*), 2.5 mM ADP, 50 mM KCl, 5 mM MgCl2,0.15 mM NADH, 0.5 U LDH, and 1-10 µl

Table 1. Effect of exercise on the activities (units/g wet weight) of glycolytic andassociated enzymes in white muscle of horse mackerel.


HK 0.28 ± 0.09 0.25 ± 0.06 0.17 ± 0.02PGI 14.5 ± 0.4 15.6 ± 0.7 13.9 ± 0.2PFK 0.76 ± 0.03 0.55 ± 0.08a 0.36 ± 0.04a,b

Ald 52.4 ± 8.7 54.3 ± 4.5 70.4 ± 7.1PK 241 ± 24 112 ± 13a 88.0 ± 7.7a

LDH 140 ± 4 131 ± 17 121 ± 9PGM 3.01 ± 0.16 2.41 ± 0.15 2.01 ± 0.32a

FBPase 0.57 ± 0.04 0.43 ± 0.05a 0.21 ± 0.01a,b

Data are reported as means ± SEM of 3-4 determinations in tissue from separateindividuals. aP<0.05 compared to the corresponding control value and bP<0.05 com-pared to the 5-min burst exercise group (two-tailed Dunnett test).HK, hexokinase; PGI, phosphoglucoisomerase; PFK, phosphofructokinase; Ald, aldo-lase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PGM, phosphoglucomutase;FBPase, fructose-1,6-bisphosphatase.

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values. FBPase activity was also reducedafter 60-min cruiser swimming to 33% of thecontrol value. By contrast, PFK activity roseby 11-fold and LDH by 1.8-fold after 5-minburst exercise. However, neither enzyme wasaffected by sustained 60-min swimming. Inliver, stimulation of swimming affected onlyPGI and PK during 5-min burst swimming(Table 3). PGI activity fell to 59% of thecontrol value whereas PK activity rose by2.3-fold. No enzymes were affected duringsustained cruiser swimming. In brain, Aldactivity increased by 62% during both formsof exercise, whereas PGM activity wassharply reduced to 32% of control after 60min sustained exercise and was undetectablein the tissue from fish submitted to 5-minburst exercise (Table 4). FBPase was alsoundetectable in brain of exercised fish.

Figures 1-4 show the distribution of en-zymes between free and bound forms inhorse mackerel tissues. In white muscle,swimming exercise led to the redistributionof six of eight enzymes, but did not affectPGI or PGM (Figure 1). Short-term burstswimming significantly reduced the percent-age of bound PFK, Ald and FBPase andreleased all HK into the free fraction. Incontrast, bound PK rose from about 15 to54% of the total and the percentage of boundLDH also doubled. Sustained cruiser swim-ming had different effects on the distributionof enzymes in white muscle. Although PFKand PK responded similarly to both forms ofexercise, the effect on FBPase was muchmore pronounced with no bound FBPasefound in white muscle during this type ofswimming. HK, Ald and LDH were unaf-fected during cruiser swimming.

Red muscle showed fewer changes in theproportions of bound enzymes during swim-ming exercise. The only effect of burst exer-cise on this tissue was that bound LDHdropped by one-half. By contrast, sustainedcruiser exercise affected the bound propor-tion of four enzymes - PFK, PK, LDH andPGM. Bound PGM activity was not found at

Table 3. Effect of exercise on the activity (units/g wet weight) of glycolytic andassociated enzymes in liver of horse mackerel.


HK 0.58 ± 0.07 0.40 ± 0.02 0.65 ± 0.15PGI 10.8 ± 1.8 6.4 ± 0.6a 8.1 ± 0.2PFK 0.37 ± 0.32 0.32 ± 0.02 0.38 ± 0.09Ald 2.65 ± 0.22 2.46 ± 0.15 2.34 ± 0.28PK 6.9 ± 0.4 16.0 ± 1.7a 8.0 ± 0.6b

LDH 1.57 ± 0.37 1.64 ± 0.18 1.26 ± 0.08PGM 3.12 ± 0.28 2.87 ± 0.37 2.31 ± 0.28G6PDH 5.63 ± 0.60 4.45 ± 0.64 3.92 ± 0.04FBPase 2.88 ± 0.26 2.43 ± 0.27 2.76 ± 0.19

Data are reported as means ± SEM of 3-4 determinations in tissue from separateindividuals. aP<0.05 compared to the corresponding control value and bP<0.05 com-pared to the 5-min burst exercise group (two-tailed Dunnett test).G6PDH, glucose-6-phosphate dehydrogenase. For other abbreviations, see legend toTable 1.

Figure 1. Effect of exercise onthe distribution of glycolytic andassociated enzymes betweenfree and bound fractions in whitemuscle of horse mackerel. Con-trol (white bars), 5-min burstswimming (grey bars) and 60-min cruiser swimming (blackbars). Data are reported asmeans ± SEM (N = 3-4 individualfishes). aP<0.05 compared to thecorresponding control value andbP<0.05 compared to the 5-minburst exercise group (two-tailedDunnett test). HK, hexokinase; PGI, phosphoglucoisomerase; PFK, phosphofructokinase;Ald, aldolase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PGM, phosphoglucomu-tase; FBP, fructose-1,6-bisphosphatase.

Table 4. Effect of exercise on the activities (units/g wet weight) of glycolytic andassociated enzymes in brain of horse mackerel.


HK 0.21 ± 0.04 0.21 ± 0.01 0.18 ± 0.01PGI 3.72 ± 0.11 3.97 ± 0.11 3.72 ± 0.11PFK 0.18 ± 0.03 0.24 ± 0.03 0.25 ± 0.01Ald 1.66 ± 0.11 2.69 ± 0.18a 2.69 ± 0.12a

PK 36.5 ± 1.8 34.5 ± 1.7 33.3 ± 1.2LDH 9.20 ± 0.43 7.49 ± 0.36 8.24 ± 0.83PGM 0.22 ± 0.06 0 0.07 ± 0.03a

G6PDH 0.07 ± 0.01 0 0.08 ± 0.02FBPase 0.05 ± 0.02 0 0

Data are reported as means ± SEM of 3-4 determinations in tissue from separateindividuals. aP<0.05 compared to the corresponding control value (two-tailed Dunnetttest).G6PDH, glucose-6-phosphate dehydrogenase. For other abbreviations, see legend toTable 1.

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about 61% after burst exercise. Sustainedexercise had the same effects as burst exer-cise on liver HK, PK and FBPase. However,the percentage of bound PGI and G6PDHdid not change during sustained swimming.The proportion of bound LDH was reducedduring cruiser swimming in liver comparedto both the control and burst exercise situa-tion and, in addition, no bound PGM wasfound in liver after sustained exercise.

In brain both exercise regimens increasedthe percentage of bound HK but PGI, Aldand LDH were unaffected by either form ofexercise (Figure 4). PFK binding was notaffected during burst exercise, but percentbinding fell from about 53% in control to11% after sustained 60-min swimming. Incontrast, the percentage of bound PK in brainincreased from about 8 to 16% during sus-tained swimming.

Discussion

The effect of exercise on intermediarymetabolism and energetics of organisms hasbeen well studied (31), including numerousstudies of metabolic responses to exercise byfish species (4,5,8). Two areas of exercisemetabolism in fish that are still not fullyresolved, however, are the effects of exer-cise on enzyme activities and on the distribu-tion of enzymes between free and boundstates. Therefore, in this study we focused onthese two phenomena, monitoring the ef-fects of burst high-speed swimming (5 minat 1.8 m/s) and of sustained moderate-speedswimming (60 min at 1.2 m/s) on the maxi-mal activities of enzymes in four organs andon the subcellular distribution of enzymesbetween free and bound states. Short-termburst exercise is typically powered by whitemuscle in fish, whereas long-term cruiserexercise is primarily a function of red muscle.Previous work from our Karadag laboratoryhas analyzed the different modes of swim-ming and their effects on metabolite levels inhorse mackerel tissues. These fish can swim

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Figure 2. Effect of exercise onthe distribution of enzymes be-tween free and bound fractionsin red muscle of horse mackerel.Other information as in the leg-end to Figure 1.

Figure 3. Effect of exercise onthe distribution of enzymes be-tween free and bound fractionsin liver of horse mackerel.G6PDH, glucose-6-phosphatedehydrogenase. Other informa-tion as in the legend to Figure 1.

Figure 4. Effect of exercise onthe distribution of enzymes be-tween free and bound fractionsin brain of horse mackerel. Otherinformation as in the legend toFigure 1.

all, bound PK activity fell compared to con-trol and bound LDH and PFK rose comparedto burst exercise in red muscle (Figure 2).

In liver, both exercise regimens led tochanges in percent binding of several en-zymes but did not affect PFK or Ald distribu-tion (Figure 3). After 5-min burst swimmingthe percentages of bound HK, PK, FBPaseand G6PDH all increased significantly,whereas bound PGI decreased. The increasein bound G6PDH was particularly large, ris-ing from only about 1% bound in controls to

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for 7-10 h at a speed of 1.2 m/s (1). After 1 hof swimming at this speed the levels of phos-phocreatine were reduced by about 50% inwhite muscle and ATP and AMP fell byabout 10-20%, whereas ADP and inorganicphosphate levels were increased about 40%.The effects of exercise on the carbohydratecontent of horse mackerel tissues were alsostudied. Glycogen concentration decreasedand lactic acid concentration increased dur-ing the first 10-30 min of cruiser swimmingin white muscle. But even though glycogenhad fallen further after 60 min of exercise,lactate concentration was reduced well be-low the initial level, suggesting a possiblelactate export from the muscle during sus-tained swimming. Red muscle responded toexercise similarly, although the rates ofchange in metabolite levels were different.

The results of the present study show thatexercise also modifies the activities of en-zymes of intermediary metabolism in horsemackerel tissues. The enzymes chosen forstudy were several glycolytic enzymes aswell as the gluconeogenic enzyme FBPase,and G6PDH, the first enzyme of the hexosemonophosphate shunt. With the exceptionof HK, all enzymes showed some exercise-related change in maximal activity in at leastone of the four tissues tested. Both burst andcruiser exercise resulted in reduced activi-ties of PFK, PK and FBPase in white musclebut a relatively constant PFK:FBPase ratiowas maintained, suggesting that exercise didnot change the relative rates of glycolyticversus gluconeogenic flux in the muscle.The mechanism(s) regulating the coordinateddecrease in the maximal activities of PFK,PK and FBPase remain(s) to be determinedbut notably these three enzymes are typicallysubject to multiple regulatory controls by avariety of factors. One of these is reversiblephosphorylation via protein kinases and phos-phatases, which is not a mechanism knownto control any of the other five enzymes.Hence, it would be interesting to determinewhether exercise stimulated changes in the

phosphorylation state of these enzymes.Exercise-induced changes in the activi-

ties of enzymes in red muscle were quitedifferent except that, as in white muscle, theactivity of FBPase decreased in exercisedmuscle. In this case, however, PFK activitydid not decrease during exercise (indeed, itrose substantially during burst exercise) sothat the net consequence would be a de-crease in the potential for gluconeogenicflux during exercise. Indeed, the PFK:FBPaseratio rose from a control value of 0.31 tovalues during burst and cruiser exercise of8.39 and 1.11, respectively, showing thatglycolysis is clearly favored during exercise.However, the very large increase in PFKactivity during burst work is surprising, sinceit occurred within 5 min. Hence, this cannotbe synthesis of new enzyme, but must repre-sent a change of the existing enzyme eitherdue to effects of very powerful allostericeffectors (whose influence is still felt whenthe homogenate is diluted about 20-fold inthe assay mixture) or to a stable change ofenzyme properties such as those induced byreversible phosphorylation, polymerizationor alteration of bound/free status, all knownmechanisms of PFK control (32). However,the results illustrated in Figure 2 indicatethat a change in the relative amounts ofbound versus free PFK could not account forthe change in enzyme activity.

Small changes in the activities of glyco-lytic enzymes in liver and especially in brainsuggest that metabolism is not perturbedvery much during exercise in these tissues.Burst work typically activates glycogenoly-sis in vertebrate liver to increase the supplyof glucose to the working muscle. This effectis typically accomplished via activation ofglycogen phosphorylase coupled with inhib-itory control on PFK that facilitates the di-version of hexose phosphates into glucoseoutput. Interestingly, in mackerel liver, adecrease in PGI activity during burst exer-cise might also suggest that some additionalcontrol may be applied at this enzyme locus

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which converts glucose-6-phosphate intofructose-6-phosphate for use by PFK.

Reversible enzyme binding to subcellu-lar structures and other macromolecules iswell known but its importance in the controlof enzymes and pathways still remains con-troversial. Previous studies from our labora-tories found that the distribution of enzymesof carbohydrate metabolism between freeand bound fractions may be modified byexercise in trout (12,29), anoxia in goldfish(28), and hypoxia in sea scorpion (30). How-ever, whereas in the two first species anactivation of glycolysis led to an increase orno change in enzyme binding, in sea scor-pion tissues the situation was more compli-cated. With increased glycolytic rate, thebound portion increased in some enzymes,decreased in some and did not change inothers (30).

Exercise significantly changed the distri-bution of various enzymes between free andparticle-bound states in horse mackerel tis-sues. In white muscle, for example, the dis-appearance of the bound form of HK duringburst work and of FBPase after 60-min exer-cise could significantly change the functionof these enzymes. The bound portion of PFKin white muscle also decreased by aboutone-half in both exercised groups. Detailedstudies have suggested that PFK bindingmay be one of the few interactions betweenglycolytic enzymes and subcellular struc-tures that have real significance in vivo. Thisis because PFK binding holds up under physi-ological conditions of ions and metabolites,whereas interactions between various otherglycolytic enzymes and F-actin (believed tobe a chief binding site for glycolytic en-zymes) frequently disappear at physiologi-cal ionic strength (17). However, the presentresults (reduced PFK binding in exercisedmuscle) are contrary to results for trout wherePFK binding increased during exercise (12,29). The reason for this contradiction is notapparent from the current data but might berelated to differences in the dependence of

white muscle from different species on phos-phagen versus anaerobic glycolysis for thesupport of muscle work. PK and LDH re-sponded in opposite ways to PFK duringexercise, the percent binding of both en-zymes rising during burst exercise and boundPK also rising during cruiser exercise. Thismay serve to localize these two terminalenzymes of glycolysis at positions that couldfacilitate sustained anaerobic glycolysis in-cluding ATP production by PK and the re-oxidation of NADH by LDH.

Contrary to white muscle, red muscleshowed very few changes in enzyme distri-bution during exercise. This type of muscleis used primarily in sustained cruiser exer-cise but enzyme binding changes duringcruiser swimming consisted of only a smalldecrease in percent PK binding and a com-plete loss of PGM binding. Burst swimmingonly changed LDH binding in this muscle.This may not be surprising as long-termcruiser swimming powered by red musclemay be primarily fueled by the oxidation oflipids and hence only small changes in gly-colytic rate may occur during this type ofexercise.

In liver, several enzymes showed in-creased percent binding during exercise. Oneof the most interesting effects may be theincrease in HK binding, which was alsofound in brain. These two findings may bediscussed from the point of view of coordi-nation of glycolysis with Krebs cycle opera-tion. It is well known that HK interacts withporin, a protein in the mitochondrial mem-brane (33,34). ATP synthesis within the mi-tochondria provides the ATP for the HKreaction and, at the same time, products ofthe HK reaction, glucose-6-phosphate andADP, are processed by glycolysis and mito-chondria in the near-mitochondrial space.Unfortunately, there is no comparable infor-mation on binding sites for other glycolyticenzymes in liver and brain but the exercise-induced increase in PK binding might alsoserve to localize the production of pyruvate,

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a substrate for aerobic oxidation, in a posi-tion that better facilitates pyruvate uptake bymitochondria.

There is no doubt that reversible bindingof glycolytic and other enzymes to cellularstructures can play a role in the precise regu-lation of metabolism. Unfortunately, to date,the mechanisms involved in this regulationare not well understood (14) and redistribu-tion of different enzymes in different direc-tions, as seen in the present study or in ourprevious work with sea scorpions (30), makesthe picture even more complicated. Suchredistribution indicates that this mechanism

may be involved in the regulation of glycoly-sis in its organs, providing space-time com-partmentation of the pathway to serve differ-ent metabolic needs. The specific enzymesaffected in different tissues could result intissue-specific changes in flux through dif-ferent pathways in a manner that serves themetabolic needs of each organ.

Acknowledgments

The authors thank N. Glibina for techni-cal assistance.

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