Glycosis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas. Rate-limiting steps and activation at fertilization.

The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas, before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.     

Carbohydrate and amino acid metabolism in fasting and aestivating African lungfish (Protopterus dolloi)

The potential importance of carbohydrates and amino acids as fuels during periods of fasting and aestivation in the African lungfish, Protopterus dolloi, were examined. No significant decreases in tissue glycogen levels were observed following 60 days of fasting or aestivation, suggesting lungfish may undergo 'glycogen sparing'. Yet glycogenolysis may be important during aestivation based on the differing responses of two flux-generating enzymes of the glycolytic pathway, hexokinase (HK) and pyruvate kinase (PK). PK is required for glycogen breakdown whereas HK is not. HK activity is significantly down-regulated in the heart and gill tissues during aestivation, while PK activity is sustained. The significant negative correlation between the activity of HK and glucose levels in the heart of aestivating lungfish suggests HK may be regulated by glucose concentrations. There was no indication of anaerobic glycolytic flux during aestivation as lactate did not accumulate in any of the tissues examined, and no significant induction of lactate dehydrogenase (LDH)activity was observed. The increase in glutamate dehydrogenase (GDH) and aspartate aminotransferase (Asp-AT) activities in the liver of aestivating P. dolloi suggests some energy may be obtained via increased aminoacid catabolism, leading to the generation of tricarboxylic acid (TCA) cycle intermediates. These findings indicate the importance of both carbohydrate and amino acid fuel stores during aestivation in aphylogenetically ancient, air-breathing fish.     

Alterations of the glycolytic pathway and adenine nucleotide state in livers of clofibrate treated rats.

The influence of clofibrate on the glycolytic pathway in liver was studied. The changes in the activity of glucokinase and hexokinase were not significant. A reduction of phosphofructokinase (p less than 0.05) and pyruvate kinase activity was found (p less than 0.0005) during clofibrate feeding. An in vitro inhibition of these enzymes could not be demonstrated by clofibrate up to a concentration of 2.5 mM. Crossover plots of glycolytic intermediates indicate that the reduced pyruvate kinase activity may influence the glycolytic pathway in vivo. Clofibrate feeding induces a lower ATP:ADP ratio, a lower adenylate energy charge and elevates AMP levels in rat liver. This may possibly stimulate the hepatic glycogenolysis and the glucose utilisation by this organ.     

Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.     

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.     

Glyconeogenic and glycogenic enzymes in chronically active and normal skeletal muscle

The chronically active (pseudomyotonic) gastrocnemius muscle in the C57B16J dy2J/dy2J mouse contains both elevated lactate and glycogen as well as fibers that have high amounts of glycogen and enhanced glyconeogenic activity. In the present study we analyze the activities of some key glyconeogenic enzymes to assess the causes of elevated muscle glycogen and to determine the pathway for glycogen synthesis from lactate. Glycogen synthase, malate dehydrogenase, phosphoenolpyruvate carboxykinase, and malic enzyme were all elevated in homogenates of the chronically active muscle. Activities of glycogen phosphorylase and fructose 1,6-bisphosphatase were decreased in whole muscle homogenates. Histochemistry demonstrated that the high-glycogen fibers were typically fast-twitch glycolytic fibers that had high glycogen synthase, glycogen phosphorylase, and malic enzyme activities. Malate dehydrogenase activity followed succinate dehydrogenase activity and did not correlate to high-glycogen fibers. Thus the high-glycogen fibers have an elevated enzymatic capacity for glycogen synthesis from lactate, and the pathway may involve use of the pyruvate kinase bypass enzymes.     

Glycolysis of sea urchin eggs : Rate-limiting steps and activation at fertilization

The amounts of glycolytic intermediates and adenine nucleotides in unfertilized Anthocidaris crassispina eggs and in fertilized eggs or embryos were measured. The determinations on unfertilized and fertilized (30 min) eggs of Pseudocentrotus depressus showed the same results. Calculation of both mass action ratios and free energy changes for each enzymatic step of glycolysis showed that reactions catalysed by α-glucan phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were rate-limiting steps of glycolysis in both unfertilized and fertilized eggs. It also suggested that these three key or rate-limiting enzymes were activated by fertilization. Phosphorylase is activated at fertilization as is also pyruvate kinase. Activation of phosphorylase is also shown by the measurement of the activity in homogenate. Phosphofructokinase showed no increase in activity until 20 min after fertilization, the increase then being closely correlated with a decline in phosphate potential. On the basis of their mass action ratios, none of these rate-limiting enzymes appears to have reached a state of equilibrium by hatching (20 h). The temporal discontinuities in the activation pattern of these three enzymes suggests that no single control mechanism can be operative during the first hour following fertilization.     

GLYCOGEN METABOLISM AND CHANGES IN THE ACTIVITIES OF PHOSPHORYLASE, PHOSPHOFRUCTOKINASE AND PYRUVATE KINASE DURING DEVELOPMENT OF SEA URCHIN EGGS

The glucose and glycogen contents of sea urchin eggs and embryos were measured enzymatically. Unfertilized eggs of Hemicentrotus pulcherrimus and Anthocidaris crassispina contain about 20.9 and 24.4 μg of glycogen per mg protein, respectively. As for glucose, unfertilized eggs of Hemicentrotus and Anthocidaris contain about 0.7 and 1.9 μg per mg protein, respectively. Glycogen consumption during embryonic development differs with different species of sea urchins. In Anthocidaris , glycogen decreases significantly after fertilization. The oxidation of glucose and glycogen accounts for about 50% of oxygen consumed until the early blastula stage in this species. The contribution ratio of glucose and glycogen to the overall energy pool becomes less than 10% at later stages. In Hemicentrotus , however, the glycogen content remains unchanged until the early blastula stage and thereafter decreases. The importance of glucose and glycogen as an energy fuel seems little throughout the development of Hemicentrotus. Activities of phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40) were measured at various embryonic stages in both species of sea urchins. The difference between two species in the consumption of glucose and glycogen can not be elucidated by the differences in the activities of these enzymes.     

Intracellular mechanism of action of sympathetic hepatic nerves on glucose and lactate balance in perfused rat liver

In rat liver perfused in situ stimulation of the nerve plexus around the hepatic artery and the portal vein caused an increase in glucose output and a shift from lactate uptake to output. The effects of nerve stimulation on some key enzymes, metabolites and effectors of carbohydrate metabolism were determined and compared to the actions of glucagon, which led to an increase not only of glucose output but also of lactate uptake. 1. Nerve stimulation caused an enhancement of the activity of glycogen phosphorylase a to 300% and a decrease of the activity of glycogen synthase I to 40%, while it left the activity of pyruvate kinase unaltered. Glucagon, similarly to nerve action, led to a strong increase of glycogen phosphorylase and to a decrease of glycogen synthase; yet in contrast to the nerve effect it lowered pyruvate kinase activity clearly. 2. Nerve stimulation increased the levels of glucose 6-phosphate and of fructose 6-phosphate to 200% and 170%, respectively; glucagon enhanced the levels to about 400% and 230%, respectively. The levels of ATP and ADP were not altered, those of AMP were increased slightly by nerve stimulation. 3. Nerve stimulation enhanced the levels of the effectors fructose 2,6-bisphosphate and cyclic AMP only slightly to 140% and 125%, respectively; glucagon lowered the level of fructose 2,6-bisphosphate to 15% and increased the level of cyclic AMP to 300%. 4. In calcium-free perfusions the metabolic responses to nerve stimulation showed normal kinetics, if calcium was re-added 3 min before, but delayed kinetics, if it was re-added 2 min after the onset of the stimulus. The delay may be due to the time required to refill intracellular calcium stores. The hemodynamic alterations dependent on extracellular calcium were normal in both cases. The activation of glycogen phosphorylase, the inhibition of glycogen synthase and the increase of glucose 6-phosphate can well explain the enhancement of glucose output following nerve stimulation. The unaltered activity of pyruvate kinase and the marginal increase of fructose 2,6-bisphosphate cannot be the cause of the nerve-stimulation-dependent shift from lactate uptake to output. The very slight increase of the level of cyclic AMP after nerve stimulation cannot elicit the observed activation of glycogen phosphorylase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phosphocreatine production coupled to the glycolytic reactions in the cytosol of cardiac cells

Phosphocreatine production catalyzed by a cytosolic fraction from cardiac muscle containing all glycolytic enzymes and creatine kinase in a soluble form has been studied in the presence of creatine, adenine nucleotides and different glycolytic intermediates as substrates. Glycolytic depletion of glucose, fructose 1,6-bis(phosphate) and phosphoenolpyruvate to lactate was coupled to efficient phosphocreatine production. The molar ratio of phosphocreatine to lactate produced was close to 2.0 when fructose 1,6-bis(phosphate) was used as substrate and 1.0 with phosphoenolpyruvate. In these processes the creatine kinase reaction was not the rate-limiting step: the mass action ratio of the creatine kinase reaction was very close to its equilibrium value and the maximal rate of the forward creatine kinase reaction exceeded that of glycolytic flux by about 6-fold when fructose 1,6-bis(phosphate) was used as a substrate. Therefore, the creatine kinase raction was continuously in the state of quasiequilibrium and the efficient synthesis of phosphocreatine observed is a result of constant removal of ADP by the glycolytic system at an almost unchanged level of ATP ([ATP] ? [ADP]), this leading to a continuous shift of the creatine kinase equilibrium position.When phosphocreatine was added initially at concentrations of 5–15 mM the rate of the coupled creatine kinase and glycolytic reactions was very significantly inhibited due to a sharp decrease in the steady-state concentration of ADP. Therefore, under conditions of effective phosphocreatine production in heart mitochondria, which maintain a high phosphocreatine: creatine ratio in the myoplasm in vivo, the glycolytic flux may be suppressed due to limited availability of ADP restricted by the creatine kinase system. The possible physiological role of the control of the glycolytic flux by the creatine kinase system is discussed.     

Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes

This study investigates the effect of overexpression of key glycolytic enzymes exhibiting either native or alternative allosteric regulation on glucose bioconversion by resting Escherichia coli cells previously engineered for ethanol production. Homologous and heterologous pyruvate kinases (Pyk) and phosphofructokinases (Pfk) were individually and simultaneously overexpressed. Overexpression of the E. coli Pfk led to a shift from ethanol to lactate formation (three-fold above the control level) while overexpression of Pyks accelerated lactate formation two-fold with less reduction in ethanol formation. Further increase in lactate formation (five-fold above the control level) resulted from overexpression of Pfk from Lactobacillus bulgaricus which, unlike the E. coli Pfk, is not allosterically regulated by either phosphoenolpyruvate or ADP. These effects on the carbon flux distribution were accompanied by significant changes in the intracellular concentrations of several glycolytic intermediates. Increased Pfk levels led primarily to reduced levels of hexose phosphates. Increased Pyk activity resulted in more complex changes which were different for overexpressed native Pyk and for overexpressed Bacillus stearothermophilus Pyk, which differs from E. coli Pyk in lacking activation by fructose 1,6-diphosphate, but is allosterically activated by AMP and ribose 5-phosphate. Simultaneous overexpression of native Pfk and Pyk caused a Pfk-overexpression-like phenotype with lower levels of hexose phosphates and further increased lactate formation (nine-fold above the control level). The flux data demonstrate that overexpression of even single enzymes early in a central pathway can increase the fluxes to a particular metabolic product, although it may not affect the glucose uptake rate.     

Mechanistic model of cardiac energy metabolism predicts localization of glycolysis to cytosolic subdomain during ischemia

A new multidomain mathematical model of cardiac cellular metabolism was developed to simulate metabolic responses to reduced myocardial blood flow. The model is based on mass balances and reaction kinetics that describe transport and metabolic processes of 31 key chemical species in cardiac tissue. The model has three distinct domains (blood, cytosol, and mitochondria) with interdomain transport of chemical species. In addition to distinguishing between cytosol and mitochondria, the model includes a subdomain in the cytosol to account for glycolytic metabolic channeling. Myocardial ischemia was induced by a 60% reduction in coronary blood flow, and model simulations were compared with experimental data from anesthetized pigs. Simulations with a previous model without compartmentation showed a slow activation of glycogen breakdown and delayed lactate production compared with experimental results. The addition of a subdomain for glycolysis resulted in simulations showing faster rates of glycogen breakdown and lactate production that closely matched in vivo experimental data. The dynamics of redox (NADH/NAD+) and phosphorylation (ADP/ATP) states were also simulated. These controllers are coupled to energy transfer reactions and play key regulatory roles in the cytosol and mitochondria. Simulations showed a similar dynamic response of the mitochondrial redox state and the rate of pyruvate oxidation during ischemia. In contrast, the cytosolic redox state displayed a time response similar to that of lactate production. In conclusion, this novel mechanistic model effectively predicted the rapid activation of glycogen breakdown and lactate production at the onset of ischemia and supports the concept of localization of glycolysis to a subdomain of the cytosol.     

Regulation of cleavage by protein kinase C in Chaetopterus

We report that protein kinase C (PKC) plays a regulatory role in early cleavage in Chaetopterus eggs. Using Western blotting, we assayed the expression patterns of conventional PKCs (cPKC), novel PKCs (nPKC), and atypical PKCs (aPKC). During early development after fertilization, PKC protein levels varied independently by isoform. PKC protein expression during differentiation, without cleavage and after parthenogenetic activation, was very similar to that during normal development indicating that PKC gene expression does not require cellularization. Since PKC has been shown to regulate meiosis in this organism, we also assayed the membrane association of these isoforms as an indicator of their activation during meiosis and early cleavage. PKC-gamma transiently associated with membranes and therefore became activated before meiotic division and cleavage, whereas PKC-alpha and -beta transiently dissociated from membranes and therefore became inactivated at these times. Inhibition of these PKC isoforms by bisindolylmaleimide I had no effect on cleavage or early development to the trochophore larva, indicating that PKC-gamma activation is not essential for cleavage or early development. However, their persistent activation by thymeleatoxin blocked cleavage. The results indicate that the dissociation of PKC-alpha and/or -beta from the membrane fraction, and therefore their inactivation, is essential for normal cleavage. Elevated PKC activity is essential for nuclear envelope breakdown and spindle formation at meiosis I. By contrast, down-regulation of this activity is essential for cleavage after fertilization.     

Distribution of some enzymes concerning carbohydrate metabolism in sea urchin eggs

As an aid to the elucidation of the mechanism of activation of glycolysis upon fertilization, the activity and the distribution of the enzymes concerned were measured in unfertilized and fertilized eggs of Hemicentrotus pulcherrimus and Pseudocentrotus depressus. The enzymes investigated were phosphorylase, exo-1,4-α-glucosidase, hexokinase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, 6-phosphofructokinase, hexosediphosphatase, fructose-bisphosphate aldolase, pyruvate kinase, and lactate dehydrogenase.Phosphorylase and pyruvate kinase were the enzymes which were activated upon fertilization. Glucose-6-phosphate dehydrogenase and a part of aldolase changed their distribution from the particulate to the soluble fraction upon fertilization. Advantages of enzyme activation over changes in enzyme distribution upon fertilization were discussed as a mechanism for the fertilization-induced activation of glycolysis.     

Control of glycolysis in the posterior adductor muscle of the sea musselMytilus edulis

Summary Concentrations of glycolytic intermediates, lactate, adenine nucleotides, inorganic phosphate, phosphoarginine and citrate have been estimated after various periods of valve closure (Table 1 and Fig. 1). Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals glycogen phosphorylase, phosphofructokinase, hexosediphosphatase and pyruvate kinase catalyze non-equilibrium reactions. The first three enzymes possess relatively low activities (Table 2).From the changes in concentrations of the glycolytic intermediates it is concluded that phosphofructokinase controls the carbon flow during the first hours after valve closure, whereas later on the rate of conversion of phosphoenolpyruvate is determining this flow. In skeletal muscle phosphofructokinase controls the carbon flow during the whole period of exercise.The concentrations of ADP, AMP and inorganic phosphate increase, whereas the concentrations of ATP, phosphoarginine and citrate decrease during valve closure (Table 1 and Fig. 2). In contrast to skeletal muscle, these changes do not result in a strong increase in the glycolytic flux.There is a much greater potential for ATP hydrolysis by the myofibrillar ATPase system than is actually realized by the adductor muscle during valve closure.     

Small peptides released from muscle glycolytic enzymes during dry-cured ham processing

Glycolytic enzymes are a group of sarcoplasmic enzymes responsible for the extraction of the energy available from carbohydrates. The glycolytic pathway consists of 10 enzyme-catalyzed steps. Fragments identified in this study, within the range 1100-2600 Da, correspond to glycogen phosphorylase enzyme, which catalyzes the rate limiting step in the degradation of glycogen, enzymes that catalyze steps 6-10 of glycolysis (glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase, respectively), and lactate dehydrogenase, which catalyzes the interconversion of pyruvate and lactate. A total of 45 specific fragments of these enzymes resulting from the processing of dry-cured ham are reported for the first time in this work. This study evidences the intense proteolysis occurring in the sarcoplasmic fraction of dry-cured ham as well as facilitates the choice of the most adequate tools in the identification of naturally generated peptides through comparison between Paragon and Mascot search engines, together with UniProt and NCBInr databases.     

The muscle-specific calmodulin-dependent protein kinase assembles with the glycolytic enzyme complex at the sarcoplasmic reticulum and modulates the activity of glyceraldehyde-3-phosphate dehydrogenase in a Ca2+/calmodulin-dependent manner

The skeletal muscle specific Ca(2)+/calmodulin-dependent protein kinase (CaMKIIbeta(M)) is localized to the sarcoplasmic reticulum (SR) by an anchoring protein, alphaKAP, but its function remains to be defined. Protein interactions of CaMKIIbeta(M) indicated that it exists in complex with enzymes involved in glycolysis at the SR membrane. The kinase was found to complex with glycogen phosphorylase, glycogen debranching enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and creatine kinase in the SR membrane. CaMKIIbeta(M) was also found to assemble with aldolase A, GAPDH, enolase, lactate dehydrogenase, creatine kinase, pyruvate kinase, and phosphorylase b kinase from the cytosolic fraction. The interacting proteins were substrates of CaMKIIbeta(M), and their phosphorylation was enhanced in a Ca(2+)- and calmodulin (CaM)-dependent manner. The CaMKIIbeta(M) could directly phosphorylate GAPDH and markedly increase ( approximately 3.4-fold) its activity in a Ca(2+)/CaM-dependent manner. These data suggest that the muscle CaMKIIbeta(M) isoform may serve to assemble the glycogen-mobilizing and glycolytic enzymes at the SR membrane and specifically modulate the activity of GAPDH in response to calcium signaling. Thus, the activation of CaMKIIbeta(M) in response to calcium signaling would serve to modulate GAPDH and thereby ATP and NADH levels at the SR membrane, which in turn will regulate calcium transport processes.     

The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in muscles from vertebrates and invertebrates

1. The activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase in crude homogenates of vertebrate and invertebrate muscles are reported. 2. Pyruvate carboxylase activity was present in all insect flight muscles that were investigated: in homogenates of bumble-bee flight muscle the activity was inhibited by ADP and activated by acetyl-CoA, and it was distributed mainly in the mitochondrial fraction. This is the first demonstration of pyruvate carboxylase activity in muscle. However, the activity appears to be restricted to insect flight muscle, since it was not found in other invertebrate or vertebrate muscles. 3. Since the three enzymes were never found together in the same muscle, it is concluded that these enzymes cannot provide a pathway for the synthesis of glycogen from lactate or pyruvate in muscle. Other roles for these enzymes in muscle are suggested. In particular, pyruvate carboxylase may be present in insect flight muscle for the provision of oxaloacetate to support the large increase in activity of the tricarboxylic acid cycle which occurs when an insect takes flight.     

Glycolysis in the eye tissues of the rabbit in ontogeny. I. The enzymes of glycolysis and hexosemonophosphate shunt]

The activity of the enzymes of glycolysis (phosphofructokinase, aldolase, pyruvate kinase, lactate dehydrogenase) and hexose monophosphate shunt (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) was determined in the eye tissues of the rabbit at different stages of ontogenesis. The activity of these enzymes in the retina was shown to be higher than in other eye tissues. In the uveal tract (iris, ciliary bodies, uvea) the activity of glycolytic enzymes changes with the age. The greatest changes in the activity of enzymes were found during the period of the opening of eyelids. The activity of the enzymes of hexose monophosphate shunt in the eye tissues increases with the age. The relative activity of dehydrogenases of the hexose monophosphate shunt after the establishment of visual function is, however, not high and does not exceed that of phosphofructokinase and pyruvate kinase in the eye tissues of the rabbit.