Regulation Of Glycolysis In Muscle Research Articles

Regulation of rainbow trout white muscle phosphofructokinase during exercise

1. 1. Studies of purified 6-phosphofructo-1-kinase (PFK) from white skeletal muscle of the rainbow trout ( Oncorhynchus my kiss) were undertaken to illuminate aspects of the regulation of glycolysis in muscle under resting versus exercising conditions. 2. 2. Trout muscle PFK was strongly inhibited by ATP at physiological levels and was regulated by a number of allosteric effectors. 3. 3. Allosteric activators included ammonium ion, inorganic phosphate, AMP, ADP, and fructose-2,6-bisphosphate (F2.6P2); these enhanced enzyme affinity for F6P and reversed inhibition by ATP. 4. 4. Changes in pH also played a major role in PFK regulation; as pH decreased from 7.2 to 6.6 (mimicking the pH decrease during exhaustive exercise), not only was enzyme activity reduced, but the reaction cooperativity increased as well. 5. 5. The negative effect of reduced pH, however, was fully compensated for by the rise in the levels of positive modulators, such AMP following exercise. 6. 6. When assayed under substrate and effector concentrations that reflected “resting” vs “exercised” situations in muscle, the S 0.5 values for F6P were reduced by 50- and 123-fold, respectively, to values of 0.068 and 0.15 mM, both close to the physiological levels of F6P.

Regulation of glycolytic flux in an energetically controlled cell-free system: The effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F 1) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 m m) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 m m), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μ m. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria ( E. J. Davis, and L. Lumeng, 1975), J. Biol. Chem. 250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.

Comparative studies of vertebrate and invertebrate pyruvate kinases

1. 1. Electrophoretic patterns showed that among the vertebrates studied, L isozyme was present in rat and frog liver and also in rat kidney. 2. 2. M 2 is a frequent component in vertebrate tissues, giving support to the proposal that M 2 is the ancestral form differentiating to other isozymes. 3. 3. The above trend cannot simply apply to invertebrates. 4. 4. Muscle pyruvate kinases from various animals were inhibited by the particular phosphagen present in their muscle. 5. 5. The inhibition may have an important role in the regulation of glycolysis in muscle.

Regulation of glycolysis in muscle: The role of ammonium and synergism among the positive effectors of phosphofructokinase

It is observed that the decrease in the energy charge, increase in P i, NH 4 +, and fructose-6-phosphate observed in stimulated frog muscle act synergistically in increasing the activity of rabbit muscle phosphofructokinase 300-fold over its activity observed at the concentrations of above effector substrates found in the muscle at rest. The activity of phosphofructokinase at various concentrations of Mg 2+ and various fixed concentrations of NH 4 +, at levels of energy charge and P i corresponding to the resting and stimulated muscle were also studied. These results suggest that variations in the concentrations of effectors of phosphofructokinase resulting from contraction of muscle are responsible for the increase in the activity of enzyme in stimulated muscle and that this activation may not necessarily be geared to the contractile process itself as postulated by Karpatkin el al.

Regulation of the phosphorylase activating pathway in intact cardiac and skeletal muscle

Regulation of the phosphorylase activating pathway in striated muscles may take place at several sites. In heart there appear to be at least two receptor sites for adenyl cyclase activation: one for epinephrine, which was blocked by β-adrenergic blocking agents, and one sensitive to glucagon. The catalytic activity of phosphorylase kinase is probably dependent upon the availability of free intracellular calcium ion. After removal of calcium from the perfusion medium of rat hearts, epinephrine caused cyclic AMP formation, but not b to a transformation. Excess calcium produced b to a conversion in the absence of epinephrine. Small doses of this amine produced activation of kinase in intact dog hearts without b to a conversion again suggesting a requirement for an additional factor in the control of kinase activity. Regulation of phosphorylase activation by calcium is also suggested by the experiments of Drummond et al. (10) in which electrical stimulation of skeletal muscle produced rapid formation of phosphorylase a without either an increase in the concentration of cyclic AMP or activation of phosphorylase kinase. Thus the release of intracellular calcium ion by a drug, hormone, or consequent to the depolarization of the muscle fiber membrane, may provide a second mechanism of regulation of the activation of the phosphorylase system. The effectiveness of phosphorylase a in catalyzing glycogenolysis may be subject to further regulation in intact heart muscle. Maximal formation of phosphorylase a by epinephrine caused no net decrease in glycogen in intact dog heart as long as the concentration of high energy phosphate compounds and inorganic phosphate did not vary from controls. On the other hand, anoxia was a potent and rapid stimulus for glycogenolysis in the presence of rapid hydrolysis of high energy phosphates. Thus, under certain conditions glycogenolysis may be regulated by alterations in muscle metabolites. Intracellular compartmentalization may also play a role in the regulation of the system. Phosphorylation of the two phosphoproteins, phosphorylase kinase and phosphorylase a, appears to involve separate pools of muscle ATP. The experiments discussed in this paper suggest that the phosphorylase activating pathway can be stimulated and controlled at several points. This conclusion is in agreement with the experiments and hypotheses on the regulation of glycolysis in muscle presented by Helmreich and Cori in an earlier volume of this series of symposia (11).

Hexosephosphate formation and the regulation of glycolysis in muscle

The steady state concentrations of the hexosephosphates, and tracer data with 32P i suggest that the formation of glucose-6-phosphate in resting muscle takes place predominantly from the glucose and P i present in the extracellular phase, and that the phosphorylase system is essentially inactive under these conditions. The rate of glycogenolysis in tetanic contraction is such that it is adequate to support the very high rate of lactic acid formation under these extreme conditions. The apparent primary rate-limiting step in glycolysis is the phosphohexose isomerase reaction, both in resting muscle and in contraction. The activity of phosphofructokinase in resting muscle is sufficient so that this reaction is not rate-limiting; however, the activation of this enzyme in tetanic contraction is not necessarily adequate to the situation, and this reaction may then become rate-limiting. The tracer data indicate that the formation of fructose-1,6-diphosphate in contraction may take place by some mechanism other than the phosphofructokinase reaction.

Regulation of glycolysis in muscle

The question was raised, what causes the sudden increase in the rate of glycolysis when muscle contracts and the equally rapid decrease in rate when the muscle relaxes. For the purpose of this study, isolated frog sartorii were stimulated electrically while immersed in anaerobic Ringer's solution without glucose. The rate was varied between 3 and 48 shocks per min and the duration was 30 min. Under these conditions, the conversion of glycogen to lactate is the main energy producing reaction. The results indicated that two reactions are of prime importance in the regulation of glycolysis in the working muscle: the formation of glucose-1-P from glycogen and inorganic P through the action of the phosphorylase system and the removal of fructose-6-P through the phosphofructokinase reaction. From the relation of the concentration of hexosemonophosphates to the flow rate over the glycolytic system it was concluded that during stimulation these two enzyme systems increase their activity synchronously and proportionately. This is in contrast to the action of epinephrine which causes a much greater increase in phosphorylase activity than in phosphofructokinase activity and thereby leads to a large accumulation of hexosemonophosphates. From measurements of tissue concentration and rate of efflux of lactate from muscle immediately after cessation of stimulation it was concluded that lactate formation returned to the resting rate within 5 min. On the basis of measurements of the tissue concentrations of ATP, ADP, AMP, and P i it was concluded that changes in the concentrations of these compounds in stimulated muscle were too small to permit the increase in enzymatic rates actually observed in vivo. This applies particularly to the phosphorylase reaction for which in vitro data are available which permit predictions of rates at different concentrations of the above reactants. The concentrations of glucose-6-P, fructose-6-P and fructose-1,6-di-P were likewise not well correlated to the rate increase. The only two compounds that increased proportionately with the frequency of stimulation were end products of anaerobic glycolysis in muscle, namely lactate and α- l-glycero-P. A detailed investigation of the phosphorylase b [ rlhar2] a interconversion in stimulated muscle suggested that the phosphorylase b kinase underwent an activation-inactivation cycle closely connected with the contraction and relaxation phases of muscle. An analysis of results obtained with a strain of mice incapable of forming phosphorylase a during stimulation indicated that this interconversion, while not essential for lactate formation per se, was of importance for the time of onset and the final speed of lactate formation. A hypothesis based on localization and structural organization of the glycolytic chain in muscle is proposed which could explain the results summarized above. A mechanism is suggested which is similar in nature to the activation and inactivation of actomyosin (and perhaps of phosphorylase b kinase) by Ca ++ ions. It contains the idea of access of reactants to the catalyst during contraction and separation from reactants during relaxation.

Regulation of Glycolysis in Muscle: II. EFFECT OF STIMULATION AND EPINEPHRINE IN ISOLATED FROG SARTORIUS MUSCLE

Regulation of Glycolysis in Muscle: II. EFFECT OF STIMULATION AND EPINEPHRINE IN ISOLATED FROG SARTORIUS MUSCLE

Regulation of Glycolysis in Muscle: I. THE CONVERSION OF PHOSPHORYLASE b TO PHOSPHORYLASE a IN FROG SARTORIUS MUSCLE

SUMMARY The interconversion of glycogen phosphorylase b ti phos- phorylase a in frog sartorii contracting isotonically at 20” has been studied. When repetitive stimuli were applied, there was a lag period followed by the appearance of phosphorylase a, which increased in amount until a steady state was reached. Increasing the rate of stimulation shortened lag period and resulted in higher steady state levels of phosphorylase a. Pretreatment of muscles with epinephrine, amounts that increased the resting level of phosphorylase a only slightly, abolished the lag period completely. Changes in external work affected neither the lag period nor the steady state levels of phosphorylase a. Lowering the transmembrane potential by increasing the concentration of K+ in the external media was associated with increases in phosphorylase a even after relaxation of the initial contracture. Caffeine (lop2 M),

Regulation of Glycolysis in Muscle: III. INFLUENCE OF INSULIN, EPINEPHRINE, AND CONTRACTION ON PHOSPHOFRUCTOKINASE ACTIVITY IN FROG SKELETAL MUSCLE

Regulation of Glycolysis in Muscle: III. INFLUENCE OF INSULIN, EPINEPHRINE, AND CONTRACTION ON PHOSPHOFRUCTOKINASE ACTIVITY IN FROG SKELETAL MUSCLE