Myofibril ATPase Research Articles

The effect of low ATP concentrations on relaxation in the myosin regulated myofibrils from scallop.

Troponin-tropomyosin-regulated myofibrils show a significant increase in ATPase activity and contract in the absence of calcium when the ATP concentration falls significantly below the saturation level. By contrast, the ATPase of the myosin-regulated myofibrils of scallop striated muscle was not activated in the absence of calcium when the ATP concentration was lowered to 10mM. Nevertheless, a very small fraction of crossbridges were active at 10mM ATP resulting in very slow myofibrillar shortening. In contrast to the behaviour of rabbit contractile proteins there was no correlation between myofibrillar shortening and ATP induced turbidity changes of actomyosin taken from scallop.

P-301 - Effect of trifluoperazine on the Ca2+-sensitive actomyosin ATPase of cardiac and skeletal myofibrils

P-301 - Effect of trifluoperazine on the Ca2+-sensitive actomyosin ATPase of cardiac and skeletal myofibrils

Effects of training on biochemical and functional properties of rodent neonatal heart

This study was undertaken to determine biochemical and functional (in vivo) adaptations of the rodent neonatal heart in response to a training program of endurance running. Ten day-old rats were progressively trained on a treadmill (final intensity, 21 m/min, 30% grade, 1 h/day) until 75 days of age. The training program induced 14, 57, and 24% increases in relative heart mass, skeletal muscle citrate synthase activity, and whole-body maximal O2 uptake, respectively (P less than 0.05). Cardiac myosin (ATPase) and Ca2+-regulated myofibril ATPase were both reduced by approximately 15% in trained vs. sedentary animals (P less than 0.05). In the majority of trained hearts examined, the myosin isozyme profile reflected an estimated 14 +/- 3% shift toward the V3 or low ATPase isozyme. Left ventricular functional indices during submaximal exercise, derived from a fluid-filled indwelling cannula, indicated that the trained animals maintained similar left ventricular (LV) systolic pressure, LV + the time derivative of pressure, and systemic arterial mean blood pressure compared with their sedentary counterparts. These functional parameters were maintained even though the trained animals performed with lower submaximal exercise heart rate. These findings suggest that maximal exercise capacity can be enhanced in neonatal rats even though the biochemical potential for ATP degradation in the cardiac contractile system is lowered. We speculate that the trend to maintain the myosin isozyme pattern further in the direction of the V3 isozyme in the trained neonatal rat heart may reflect a means to economize cross-bridge cycling while maintaining normal levels of ventricle performance at a given submaximal work load.

Inhibition of tension development and actomyosin ATPase activity in barnacle muscle by the Ca2+-indicator dye antipyrylazo III

We have investigated the effects of the Ca2+-indicator dye antipyrylazo III on: (1) tension development in myofibrillar preparations from barnacle depressor muscle; and (2) actomyosin ATPase activity in myofibrils/native actomyosin isolated from barnacle muscle and in actomyosin hybrids prepared from pure (unregulated) rabbit F-actin and purified barnacle myosin. In all solutions, pCa was either heavily buffered with suitable [CaEGTA]/[EGTA] and/or measured with a calcium-electrode so as to offset the appreciable Ca2+-buffering effects of the dyes. Antipyrylazo III produced a rapid, reversible and concentration-dependent inhibition of: (1) tension development by isolated barnacle myofibrils; (2) calcium-regulated ATPase activity in barnacle myofibrils and native actomyosin; and (3) calcium-independent actin-activated ATPase activity in hybrid actomyosins prepared from purified barnacle myosin and rabbit actin. This latter observation indicated that the inhibitory effect of the dye on calcium-regulated tension and ATPase in intact myofibrils is due to specific repression of active crossbridge formation rather than modification of calcium-regulatory mechanisms. The hypothesis that antipyrylazo III specifically represses active crossbridge formation was supported by the observation that the dye had no effect on rigor tension development. Specific and saturable binding of the dye to these myofibrils was characterized by a maximum capacity of 1.3 mumol dye g-1 myofibrillar protein, consistent with a calculated 1 dye: 1 myosin stoichiometry. These various biological effects were observed with both commercially available antipyrylazo III and highly purified dye preparations. Preliminary studies using myofibrillar preparations from rabbit psoas muscle, guinea-pig portal vein smooth muscle, and scallop adductor muscle have indicated that contractile function in these muscle types does not appear to be inhibited by antipyrylazo III.

Function of M-line-bound creatine kinase as intramyofibrillar ATP regenerator at the receiving end of the phosphorylcreatine shuttle in muscle.

After 10 wash cycles, 0.8 u.e. of creatine kinase activity remained bound per mg of chicken pectoralis myofibrils which had been freed of soluble creatine kinase, mitochondria, and membranes. The bound creatine kinase is located at the M-band and contributes to the electron density of this sarcomeric structure (Wallimann, T., Pelloni, G.W., Turner, D.C., and Eppenberger, H. M. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 4296-4300). By measuring the combined actin-activated Mg2+-ATPase and creatine kinase reactions of myofibrils by pH-stat, it was shown that the amount of M-line-bound creatine kinase activity was sufficient to rephosphorylate the ATP hydrolyzed in vitro by the actin-activated Mg2+-ATPase. The amount of M-line-bound creatine kinase and thus the ATP regeneration potential depended on the muscle type. It was higher in fast muscles and lower in slow muscles. Inhibition of myofibrillar creatine kinase or extraction of the M-line-bound enzyme abolished the ATP regeneration potential without affecting ATPase activity. Inhibitors of myokinase, mitochondrial ADP/ATP translocase, and respiration did not affect the ATP regeneration potential or the ATPase. M-line-bound creatine kinase, sufficient to support an ATP turnover rate of 6s-1 per myosin head, seems to have the capacity for the intramyofibrillar regeneration of most or all of the ATP hydrolyzed by the myofibrillar ATPase during muscle contraction. Thus, M-line-bound creatine kinase at the myofibrillar receiving end of the phosphorylcreatine shuttle is of physiological significance.

Myofibril atpase activity of cardiac and skeletal muscle of exhaustively exercised rats

1. 1. The activation characteristics of Mg-ATP and Ca 2+ on cardiac and skeletal muscle myofibril ATPase activity were studied in rats following a run to exhaustion. In addition, the effect of varying ionic strength was determined on skeletal muscle from exhausted animals. The exhausted group (E) ran at a speed of 25 m min −1 with an 8% incline. 2. 2. Myofibril ATPase activities for control (C) and E were determined with 1, 3 and 5 mM Mg-ATP and 1 and 10 μM Ca 2+ at pH 7.0 and 30°C. 3. 3. For control skeletal muscle, at 1 and 10μM Ca 2+, there was an increase in ATPase activity from 1 to 5 mM Mg-ATP ( P < 0.05). 4. 4. For E animals the myofibril ATPase activities at 10 μM Ca 2+ and all Mg-ATP concentrations were similar to C ( P > 0.05). At 1.0 μM Ca 2+ and all Mg-ATP concentrations were similar to C ( P > 0.05). At 1.0 μM Ca 2+ the activities at 3 and 5mM Mg-ATP were greater for the E animals ( P <0.05). 5. 5. Increasing KC1 concentrations resulted in greater inhibition for E animals. 6. 6. With cardiac muscle, the myofibril ATPase activities at 1.0 μM free Ca 2+ were lower for E at all Mg-ATP levels ( P < 0.05). In contrast, at 10 μM Ca 2+, the E group exhibited an elevated myofibril ATPase activity. 7. 7. The results indicate that Mg-ATP and Ca 2+ activation of cardiac and skeletal muscle myofibril ATPase is altered with exhaustive exercise.

Calcium requirements of cardiac myofibril atpase activity following e8haustive exercise

Myocardial contractility is reduced in rats following strenuous activity. Thus, the purpose of this study was to determine some of the cellular mechanisms that may contribute to the depressed contractile function. 1. 2. Myofibril ATPase activity was determined with varying free calcium and monomeric vanadate (V 1) concentrations. 2. 3. The Mg 2+ stimulated myofibril ATPase activities were significantly reduced in the activity group (E). 3. 4. Myofibril ATPase activity from control animals increased from 0.056 ± 0.021 to 0.216 ± 0.030 μmol -P 1-mg −1 · min −1 with 0.1–10.0μM Ca 2+. 4. 5. The addition of 15.0μM V; resulted in a 37% decrease in ATPase activity of C animals. 5. 6. With regard to the experimental group, the myofibril ATPase activity at 0.1 and 1.0μM Ca 2+ were depressed ( P < 0.05) with the values at 5.0 and 10.0μM Ca 2+ being similar to the control group ( P >0.05). 6. 7. Incubations with V i resulted in an enhanced myofibril ATPase activity for E compared to C animals. 7. 8. The ATPase activities were increased by 17, 10, 10 and 15% at 3.0, 5.0. 10.0 and 15.0μM V 1. 8. 9. The results suggest that the exhaustive exercise raises the CA 2+ requirement for half-maximal activation of cardiac myofibril ATPase activity and that the contracto-regulatory mechanism of cardiac muscle is similarly altered.

Reaction intermediates of myosin ATPase from scallop adductor muscles: nonidentical two-headed structure of striated adductor muscle myosin.

To determine whether or not the two heads of myosin from striated adductor muscles of scallop are nonidentical and the main intermediate of the ATPase reaction, MADPP, is produced only on one of the two heads, the Pi-burst size, the amount of total bound nucleotides and the amount of bound ADP during the ATPase reaction were measured in this study. The Pi-burst size was 1 mol per mol in the presence of 0.1-5 mM Mg2+ ions. The amount of total nucleotides bound to myosin was 2 mol per mol. Both the amounts of bound ADP and ATP at sufficiently high ATP concentrations were 1 mol per mol of striated adductor myosin, and the affinity for ADP binding was higher than that for ATP binding. These findings indicate that MADPP or MATP is produced on each of the two heads of striated adductor myosin on its interaction with ATP. The fluorescence intensity at 340 nm of striated adductor myosin was enhanced by about 7% upon addition of ATP. The time for the half maximum fluorescence enhancement, tau 1/2, at 5 microM ATP was 0.25 s, which was almost equal to the tau 1/2 values for the Pi-burst and for the dissociation of actomyosin reconstituted from striated adductor myosin and skeletal muscle F-actin. The dependences on ATP concentration of the extent of the fluorescence enhancement and the dissociation of actomyosin could be explained by assuming that these changes are associated with the formation of MADPP on one of the two heads of myosin. The Pi-burst size and the amount of bound ADP of smooth adductor myosin were slightly but significantly larger than 1 mol per mol. Both ATPase reactions of striated and smooth adductor myofibrils showed the substrate inhibition. The extent of substrate inhibition of ATPase of smooth adductor myofibrils was less than that of striated adductor myofibrils. All the present findings support the view that the nonidentical two-headed structure is required for substrate inhibition of the actomyosin ATPase reaction.

Exercise and cardiac performance capacity in rats with partial sympathectomy.

This study was undertaken to examine maximal oxygen uptake (VO2 max) and heart rate (HR) during exercise, in situ derived cardiac contractile capacity, and Ca2+ regulation of cardiac myofibril ATPase in normal female rats and in rats with partial sympathectomy (SYMX) induced by chronic injections of guanethidine. Also, normal control (NC) rats were studied during exercise with and without the beta 1-blocking drug atenolol. The results show that SYMX did not have any effect on Ca2+-regulated cardiac myofibril ATPase activity. Also similar cardiac functional capacity, as reflected in left ventricular pressure development and +dP/dtmax, was observed for both groups when the heart was stressed by either exogenous norepinephrine or maximal afterload (aortic constriction). Exercise VO2 max was reduced by 15% (P less than 0.05) in the SYMX compared to the NC group. Although this reduction in VO2 max corresponded to a parallel reduction in HRmax, NC rats with cardiac beta-blockade attained normal VO2 max even though their exercise HRmax was below the SYMX group. These findings suggest that normal rats with attenuated HR and contractility have a better capacity to acutely maintain VO2 max during exercise than rats with chemical sympathectomy.

Effects of physical training and thyroxine on rodent cardiac functional and biochemical properties.

The effects of 8 weeks of interval running and of 14 days of thyroxine treatment were studied on selected cardiac functional and biochemical parameters of contraction in both female guinea pigs and rats. Relative to their respective sendentary-control groups, both trained groups demonstrated typical exercise adaptations of resting bradycardia and of increases in skeletal muscle oxidative enzymes and relative heart mass (P less than 0.05). Myosin ATPase, Ca2+ regulated myofibril ATPase, and + dP/dt max were at least two-fold higher in rats compared to guinea pigs (P less than 0.001). Physical training did not augment any of these parameters in either species under the experimental conditions studied. However, biochemical and functional indices of intrinsic cardiac contractility in guinea pigs were increased by injections of thyroxine; whereas, the same treatment regimen was without effect on the rats. These findings suggest that chronic running does not alter the intrinsic cardiac contractility state in either species. Also, exercise doesn't change the thyroid state of the guinea pig to a level sufficient to affect cardiac function.

Single turnovers of adenosine 5'-triphosphate by myofibrils and actomyosin subfragment 1.

The ATPase of myofibrils has been investigated by using single-turnover methods which avoid contraction to unphysiologically short sarcomere lengths. A substoichiometric amount of ATP is mixed with myofibrils in rigor, and the rate of decay of bound ATP to ADP and Pi is followed. At 0 degree C, the rate is 0.45 s-1 and is not dependent on ionic strength in the range I = 0.035-0.175. The steady-state ATPase of myofibrils under these conditions is considerably slower (0.072 s-1) than the rate of ATP decay and must be controlled by a subsequent step. Unlabeled ATP chase experiments were used to determine the rate of release to the medium of bound ATP. This rate is comparable to that of hydrolysis and dissociation as products but is dependent on ionic strength, changing from 0.53 s-1 at I = 0.035 to 0.12 s-1 at I = 0.085. Knowledge of the rate of release of bound ATP, together with the rate of ATP binding, allows the basic equilibrium constant between the states AM + ATP and A + M*.ATP to be estimated. The value is 3 X 10(4), which corresponds to about 40% of the total basic free-energy change between physiological concentrations of medium ATP and medium ADP and Pi. Single turnovers of acto-S1 ATPase were also investigated. At 0 degree C and I = 0.02, the rate of decay of bound ATP was equal to the rate of steady-state ATPase over a range of actin concentrations up to 10 times the Km. This observation suggests that it is the same enzymatic step which controls the rates of both processes; acto-S1 models of this type are discussed.

Exercise capacity and cardiac function in trained and untrained thyroid-deficient rats

This study was undertaken to ascertain the extent that impairments in a) the oxidative capacity of skeletal muscle, b) cardiac functional and biochemical correlates of contractile capacity, and c) maximal oxygen consumption (VO2 max) can be reversed in thyroid-deficient (TD) female rats subjected to an 8-wk physical training (running) program. Compared with a normal control (NC) group, thyroid deficiency caused reductions in the following: VO2 max (-32%), skeletal muscle homogenate respiratory capacity (-50%), cardiac myosin ATPase (-58%), and in situ-derived ventricular dP/dt max (-58%) (P < 0.001). The training program restored to within normal limits skeletal muscle oxidative capacity and VO2 max, but it did not improve cardiac myosin ATPase, Ca2+ regulation of myofibril ATPase, and dP/dt max relative to TD sedentary rats. However, the heart weight-to-body weight ratio was highest among the three groups in the TD trained group. These findings suggest that maximal oxygen utilization capacity of TD rats can be normalized by physical training, even though intrinsic contractile capacity of the heart could not.

Regulatory light-chains and scallop myosin: Full dissociation, reversibility and co-operative effects

Both regulatory light-chains of scallop myosin are removed from myofibrils by a short EDTA treatment at elevated temperatures (25°C in the case of Placopecten magellanicus; 35°C in the case of Aequipecten irradians). Essential light-chains are not lost by this treatment. Denuded myofibrils readily recombine with regulatory light-chains in the presence of magnesium ions up to a stoichiometry of two moles of regulatory light-chains per mole of myosin. High-affinity, specific calcium binding sites are proportional to regulatory light-chain content and are lost upon complete removal of regulatory light-chains. Denuded myofibrils contain two non-specific divalent cation binding sites of lower affinity per myosin that are not found on intact myofibrils. Occupancy of these sites by calcium depresses the actin-activated Mg2+-ATPase activitybby about 20 to 30%. Regulatory light-chains specifically modify the actin-activated Mg2+-ATPase of scallop myofibrils, inhibit the ATPase 10 to 20-fold in the absence of calcium but increase the turnover rate by two to fourfold if calcium is present. The elevated Mg2+-ATPase of denuded myofibrils in the absence of calcium is restored biphasically by regulatory light-chains to the low rates typical of intact myofibrils. This ATPase rate remains constant until one mole of regulatory light-chain is added back, then is depressed linearly upon further re-addition of regulatory light-chains. This ATPase dependency is taken as evidence that both regulatory light-chains must be attached to intact scallop myosin for regulation, and suggests that regulatory light-chains re-add in a negatively co-operative manner.

Cooperative effects of calcium on myofibrillar ATPase of normal and hypertrophied heart.

The ATPase of myofibrils from normal and hypertrophied rat heart was studied in relation to the Ca2+-dependence of the reaction. The experimental data were fitted to theoretical curves based on the Hill equation. A positive cooperativity (n = 1.6) was found for the activation curve of the ATPase from normal heart. For hypertrophied heart no significant change in either the cooperativity or the Ca2+-sensitivity of troponin was detected. This indicates that the troponin-tropomyosin system is not altered during pressure-overload hypertrophy. However, it should be noted that no precautions were taken to control the state of phosphorylation of contractile proteins during the preparation of myofibrils.

The influence of pH on the Ca 2+-regulated ATPase of cardiac and white skeletal myofibrils

An intracellular acidosis depresses the twitch tension of cardiac muscle but in general increases that of skeletal muscle. To determine whether these contrasting effects might be due to a difference in the pH-sensitivities of the Ca 2+-activation of the myofibrils, we assayed the ATPase activity of rabbit and guinea-pig cardiac, and rabbit white skeletal, myofibrils at 37°C in solutions of different pCa (8.0 to 4.3) and pH (7.20, 7.00, 6.80, 6.40). Care was taken to free the cardiac myofibrils of contaminant membrane ATPases. Reducing the pH from 7.20 to 6.40 decreased the maximum Ca 2+-regulated portion of the cardiac myofibrillar ATPase by 20% but had no significant effect on that of the skeletal myofibrillar ATPase. This reduction of pH also produced a five-fold increase in the [Ca 2+] required for half-maximal cardiac ATPase activity at each pH; the corresponding [Ca 2+] for the skeletal myofibrillar ATPase was increased four-fold. These slight differences in the pH-sensitivities of cardiac and skeletal myofibrillar ATPases probably do not account for more than a small part of the contrasting effects of acidosis on the two types of muscle.

Kinetics and regulation of the myofibrillar adenosine triphosphatase

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+.

Adaptation of the rat myocardium to endurance training

The purpose of this study was to assess cardiac adaptation to endurance training in rats. After 11 wk of progressive treadmill exercise (1 h/day), gastrocnemius cytochrome c oxidase activity was 38% higher (P less than 0.01) in the trained (n = 20) as compared to control (n = 20) rats. Cardiac Mg2+-stimulated myofibril ATPase activity (0.308 +/- 0.012 vs. 0.324 +/- 0.006 micrometer.mg-1.min-1) did not change nor was there any change in myofibril protein concentration (60.0 +/- 1.12 vs. 59.9 +/- 0.85 mg.g-1). The isolated left ventricular papillary muscle showed no significant change in time-to-peak tension (TPT) or half-relaxation time. Tension output, however, was significantly increased with training, 2.2 +/- 0.3 vs. 1.5 +/- 0.1 g.mm-2 (P less than 0.025). Furthermore, when the papillary preparations were perfused with 0.5 mM lanthanum (La3+) to displace membrane-bound Ca2+, the time course for tension decay was significantly prolonged in the trained muscles (P less than 0.001). We conclude that endurance running of this type does not necessarily increase myofibril ATPase activity or the time course of the isometric twitch of rat papillary muscle. However, tension output per unit area does increase and this appears to be due to a greater amount of Ca2+ being made available to the contractile apparatus.

EFFECTS OF POSTMORTEM STORAGE CONDITIONS ON MYOFIBRILLAR ATPase ACTIVITY OF PORCINE RED AND WHITE SEMITENDINOSUS MUSCLE

ABSTRACTThis study was carried out to determine the effects of postmortem storage time, temperature and pH on myofibrillar proteins of red and white muscle. Myofibrils were isolated from (1) the red and white portion of semitendinosus muscle postmortem stored at 2°C and (2) at‐death red and white portions and suspended and stored in unbuffered 0.15M KC1 at 2°C and in buffered 0.15M KC1 (pH 5.5 and 7.0) at 2° and 25°C. To determine the effect of storage conditions on myofibrillar proteins, ATPase activity was assayed at different ionic strengths and with different modifiers. Assays of myofibril ATPase activity from postmortem muscle showed that (1) myofibrils from the white portion had greater ATPase activity than those from the red portion, (2) Ca2+‐modified activity from both portions increased and (3) Mg2+ ‐EGTA‐modified activity increased from the white portion, but remained unchanged from the red portion, during postmortem storage. These changes could be due to modifications of the regulatory protein components of muscle by calcium‐activated factor activity. For those myofibrils isolated from at‐death muscle and incubated under simulated storage conditions, a precipitous decrease occurred in Ca2+ ‐and Mg2+‐(low ionic strength) and Ca2+‐ and EDTA‐(high ionic strength) modified ATPase activity of myofibrils stored in 0.15M KC1, pH 5.5, at 25°C. Otherwise, little change occurred in these activities under other simulated conditions of storage (i.e., 2°, pH 5.5 and 7.0; and 25°, pH 7.0) with the exception that EGTA modified activity (indicates loss of Ca2+ sensitivity) increased from the white portion at 25° and 2°C, pH 7.0, and from the red portion at 25°C, pH 7.0. Hence, a high storage temperature of 25°C has more detrimental effect on the integrity of myofibrillar proteins, as measured by changes in ATPase activity, than does a low pH of 5.5, or fiber type.

Enzyme alterations in neonatal heart muscle during development

Time course changes in marker enzymes of respiration, glycolysis, glycogenolysis, and of myofibril ATPase were investigated in rodent neonatal heart muscle at different stages of development (day 7 to 42). Hexokinase activity increased by 25% during day 7 to 14, but it remained essentially constant thereafter. Phosphofructokinase increased by 30% during the first 4 weeks of age, and it also remained constant thereafter. Lactic dehydrogenase, which was sensitive to 10 m m pyruvate inhibition, increased in total activity by 50% over the 6 week period. On the other hand, citrate synthase and cytochrome c , markers of respiratory capacity, responded with 2.5- to 3-fold increases in levels during the 6 week period, approximating levels seen in adult hearts. Creatine phosphokinase underwent changes similar to that observed for the respiratory markers. Likewise, Ca 2+ and Mg 2+ stimulated ATPase of purified myofibrils underwent 2-fold increases during the same period of growth. These findings suggest that cardiac muscle of rodents undergoes marked changes in the major systems involved in ATP synthesis and degradation, which may play an important role in augmenting the functional potential of the heart that is known to occur with development.

Effect of functional overload on enzyme levels in different types of skeletal muscle.

The effects of bilateral functional overload on enzyme changes in fast-twitch plantaris muscles were studied on different groups of rats: 1) normal-control; 2) normal-exercise; 3) overload-control; and 4) overload-exercise. Overload was accomplished by surgical elimination of synergists. Exercising groups walked up a 65% grade, 3 m/min, 2 h/day. Peak muscle enlargement of the overload groups was reached after 5 wk. Citrate synthase, phosphofructokinase, and myofibril ATPase activities were consistantly depressed by approximately 30%, 40%, and 18%, respectively, in overload as compared to normal groups. Daily exercise prevented the decrease in only citrate synthase activity. Unilateral overload of medial gastrocnemius muscle indicated that both fast-twitch oxidative-glycogenolytic and fast-twitch glycogenolytic fiber types undergo enzyme changes in response to the functional stress. However, changes in the former were in closer agreement with the net changes seen in the plantaris than the latter. Soleus muscle responded to overload primarily with marked reductions in respiratory capacity. These findings suggest that certain enzyme systems are altered with functional overload in different fiber types. However, the alterations in certain enzyme systems may, in part, be independent of the process of hypertrophy.