Binding Of Glycolytic Enzymes Research Articles

Rapid stimulatory effect of insulin on binding of glycolytic enzymes to cytoskeleton of C-6 glial cells, and the antagonistic action of calmodulin inhibitors

Insulin was shown in our previous experiments to induce an increase in binding of glycolytic enzymes to muscle cytoskeleton. We show here the same stimulatory effect of insulin in C-6 glial cells in culture. In these cells, like in muscle, a short time of incubation with insulin (1-10 min) induced an increase in cytoskeleton bound phosphofructokinase and aldolase. This stimulatory effect of insulin could be prevented by treatment with calmodulin antagonists trifluoperazine, thioridazine or CGS 9343 B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that calmodulin is involved in this action of insulin. Our previous experiments have shown that growth factors and Ca(2+) also induce a rapid, calmodulin-mediated stimulation of binding of glycolytic enzymes to cytoskeleton. The present and previous results suggest that the rapid binding of glycolytic enzymes to cytoskeleton, may be a general mechanism, in different cells, in signal transduction of insulin, growth factors and other Ca(2+) -mobilizing hormones. The accelerated cytoskeletal glycolysis will supply local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following the binding of hormone to its receptor.

The Importance of ATPase Microenvironment in Muscle Fatigue: A Hypothesis

A broadly held opinion is that fatigue is not due to an insufficient supply of ATP to the energy consuming mechanisms because tissue [ATP] always remains at least one order of magnitude higher than Km for ATP of any ATPase. In general these findings also suggest that ATP consumption is well balanced with ATP regeneration even in the fatigued muscles. This balance is achieved by down-regulation of ATP consumption. Potentially this down-regulation could be accomplished by any product of the ATPase reaction and the role of Pi and H+ accumulation in this regulation has been discussed in the literature. The purpose of this paper is to describe known compartmentalization of ATP regeneration systems in muscle cell, their importance in the regulation of [adenine nucleotide] in the vicinity of ATPases and how such local ATP regeneration maybe important in the etiology of muscle fatigue. Available experimental evidence suggests that the binding of creatine kinase and glycolytic enzymes in the vicinity of sites where ATP is hydrolyzed and functional coupling between these ATP regenerating mechanisms and ATPase can generate ATPase microenvironments that have an important role in the regulation of ATPase function. Main function of this ATP regeneration is to keep the local ADP/ATP ratios favorable for ATPase function, which seems to be especially important when ATPase turnover is high. Unfortunately, the maximum rate of local ATP regeneration relative to that of ATP hydrolysis in vivo is not known, mainly because in vitro determinations underestimate this value due to a decrease in the fractional of loosely abound enzyme to the preparation during isolation procedure.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of erythrocyte metabolism by redox-regulated tyrosine phosphatases and kinases

We wish to elaborate a novel mechanism of metabolic regulation mediated by cytoplasmic tyrosine phosphatases and kinases. Briefly we propose that phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) bind reversibly to the N-terminus of the cytoplasmic domain of band 3. Once the enzymes are bound, they are inhibited; however, upon release they are restored to full activity. We demonstrate that control of enzyme binding and consequently control of substrate flow down the pathway is executed by phosphorylation of Tyr 8 and Tyr 21 within the glycolytic enzyme binding site at the N-terminus of band 3. This phosphorylation results in obstruction of enzyme binding, leading to enzyme activation. Importantly, the tyrosine kinase that phosphorylates band 3 is activated by oxidation, while the tyrosine phosphatase that dephosphorylates band 3 is inhibited by the same redox changes. Consequently, treatment of red cells wih oxidants such as H2O2 and ferricyanide can enhance both tyrosine phosphorylation of the N-terminus of band 3 and glycolysis in a coordinate manner. Because oxidant entry into the cell is not essential, a plasma membrane electron transport pathway is believed to mediate the oxidant's effects.

Platelet-Derived Growth Factor (PDGF) Rapidly Stimulates Binding of Glycolytic Enzymes to Muscle Cytoskeleton, Prevented by Calmodulin Antagonists

Glycolytic enzymes are known to be controlled by reversible binding to cytoskeleton. Our previous experiments have shown that insulin, epidermal growth factor (EGF), and Ca2+ induce a rapid and transient stimulation of binding of glycolytic enzymes to muscle cytoskeleton. We show here that platelet-derived growth factor (PDGF) exerts a similar action. Incubation of rat diaphragm muscle in the presence of PDGF resulted in rapid and transient stimulation of binding of phosphofructokinase (EC 2.7.11) and aldolase (EC 4.1.2.13) to muscle cytoskeleton. The increase in cytoskeleton-bound glycolytic enzymes induced by PDGF was prevented by treatment with the calmodulin antagonists trifluoperazine or CGS 9343B (a potent and selective inhibitor of calmodulin activity), which strongly suggests that Ca(2+)-calmodulin is involved in this effect of PDGF. Similarly, we previously found that stimulation of cytoskeleton-bound glycolytic enzymes exerted by insulin, EGF, or Ca2+, was also calmodulin mediated. The present and previous results suggest that the rapid, Ca(2+)-calmodulin-mediated increase in cytoskeleton-bound glycolytic enzymes, may be a general mechanism in the cell, in signal transduction of insulin, growth factors, and other Ca(2+)-mobilizing hormones. The accelerated cytoskeletal glycolysis will provide local ATP, which is required for the rapid cytoskeletal-membrane rearrangements following binding of growth factor or hormone to its receptor.

Effect of anaerobiosis and anhydrobiosis on the extent of glycolytic enzyme binding in Artermia embryos

The effect of anaerobiosis and anhydrobiosis on the extent of binding of glycolytic enzymes to the particulate fraction of the cell was studied in Artemia salina embryos. During control aerobic development, trehalase, phosphofructokinase and pyruvate kinase showed an increase in the percentage associated with the particulate fraction which is consistent with the carbohydrate-based metabolism of Artemia embryos. However, anaerobiosis resulted in decreased enzyme binding for six glycolytic enzymes; hexokinase, aldolase, pyruvate kinase and lactate dehydrogenase were the exceptions. Decreased enzyme binding was also observed after exposure to dehydrating conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms in Artemia embryos. This reversible interaction of glycolytic enzymes with structural proteins may account for part of the metabolic arrest observed during anaerobic dormancy and anhydrobiosis.

Adrenergic stimulation of glycolysis without change in glycolytic enzyme binding in rainbow trout (Oncorhynchus mykiss) erythrocytes

Whole blood from rainbow trout (Oncorhynchus mykiss) was incubated in vitro with pharmacological levels of isoproterenol. The adrenergic stimulation of glycolysis in erythrocytes (RBCs) was assessed by monitoring the rate of 14C incorporation from [6-14C]-glucose into the acid-soluble RBC fraction. During a 3-h in vitro incubation, incorporation of label into the acid-soluble RBC fraction of isoproterenol-treated whole blood (0.25 ± 0.04 μmol glucose∙mL−1 RBC∙h−1) was higher than into untreated blood (0.08 ± 0.01 μmol glucose∙mL−1 RBC∙h−1). The percentage of cell membrane binding for phosphofructokinase, aldolase, and glyceraldehyde-3-phosphate dehydrogenase ranged from 17 to 35% and was not altered by adrenergic stimulation. Adrenergic stimulation activates glycolysis in rainbow trout RBCs but not through the modulation of enzyme binding.

Uncoupling of mitochondrial oxidative phosphorylation abolishes the stimulatory action of insulin on the binding of glycolytic enzymes to muscle cytoskeleton

1. 1. We show here that treatment of diaphragm muscle with 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, abolished the stimulatory action of insulin on binding of the glycolytic enzymes, phosphofructokinase (PFK) and aldolase, to muscle cytoskeleton. This effect was demonstrated with low concentration of DNP, which caused only a small decrease in ATP and did not affect the basic levels of cytoskeleton-bound glycolytic enzymes. 2. 2. Higher concentrations of DNP, which induced a drastic decline in ATP content, caused a decrease in cytoskeleton-bound glycolytic enzymes and damage to myofibrils. 3. 3. These results suggest that mitochondrial ATP is required for both the preservation of the basal levels of cytoskeleton-bound glycolytic enzymes and cell structure, as well as for the expression of the stimulatory action of insulin on glycolytic enzymes' binding to muscle cytoskeleton.

Regulation of glycolysis via reversible enzyme binding to the membrane protein, band 3

Most metabolic pathways are regulated by feedback inhibition/activation or by covalent modification of a regulatory enzyme. In erythrocytes, however, we demonstrate that glycolysis can be modulated over 30-fold by controlling the availability of glycolytic enzyme binding sites at the extreme N terminus of the anion transporter, band 3. Direct obstruction of these inhibitory sites by anti-peptide Fab's against residues 1-15 of band 3 promotes an approximately 3-fold increase in the rate of lactate production. In contrast, enrichment of the erythrocyte cytoplasm with the band 3 peptide against which the above antibodies were raised results in a more than 10-fold decrease in the rate of lactate accumulation. Control peptides and their derived antipeptide antibodies corresponding to other sequences of band 3 or glycophorin were found to have no effect on lactate production. Analysis of changes in glycolytic intermediates during Fab treatments suggests that hexokinase may be one enzyme that is modulated by association with band 3. We conclude that the extreme N terminus of band 3 can bind and inhibit glycolytic enzymes in vivo and that it probably participates in control of red cell glycolysis.

Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions.

The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell.

Role of band 3 tyrosine phosphorylation in the regulation of erythrocyte glycolysis

Previous studies demonstrated that the in vitro tyrosine phosphorylation of the human erythrocyte anion transporter, band 3, prevented the binding of various glycolytic enzymes to the N terminus of the cytoplasmic tail. Since these enzymes are inhibited in their bound state, the functional consequences of band 3 tyrosine phosphorylation in the red cell should be to activate the enzymes and elevate glycolysis. We searched for various enhancers of band 3 tyrosine phosphorylation using a novel assay designed to measure the phosphotyrosine levels at the band 3 tyrosine phosphorylation/glycolytic enzyme-binding site. This assay measures the extent of phosphorylation of a synthetic band 3 peptide entrapped within resealed red cells. Using this assay, three distinct compounds, all mild oxidants, were found to stimulate the tyrosine phosphorylation of band 3. All three compounds were also found to elevate glycolytic rates in intact erythrocytes. Moreover, the antitumor drug adriamycin was found to coordinately prevent these agents from stimulating both band 3 tyrosine phosphorylation and erythrocyte glycolysis. These results suggest a possible function for a protein tyrosine kinase in human erythrocytes, to regulate glycolysis through the tyrosine phosphorylation of band 3.

Glycolytic and Associated Enzymes of Rainbow Trout (Oncorhynchus Mykiss) Red Cells: In Vitro And In Vivo Studies

ABSTRACT Studies were undertaken in vitro and in vivo to assess the maximal activities of 26 glycolytic and associated enzymes of rainbow trout (Oncorhynchus mykiss) red cells. The red cells possess a complete sequence of glycolytic enzymes capable of anaerobic oxidation of glucose to lactate. Red cell pyruvate kinase (PK) was inhibited by ATP (I50 ≈ 5 mmol I 1−1), but was not sensitive to alanine inhibition or fructose-1,6-bisphosphate activation. The properties of red cell PK were similar to those of the muscle-type enzyme. Lactate dehydrogenase (LDH) from the trout erythrocyte resembled LDH from trout heart in terms of pyruvate inhibition in vitro. Enzymes associated with phosphagen and amino acid metabolism as well as the pentose phosphate shunt were also present. However, enzyme indicators of glycogenolytic and gluconeogenic potential were either absent or present at very low levels. The capacity for aerobic respiration via the tricarboxylic acid (TCA) cycle was suggested by the presence of citrate synthase activity. The association of glycolytic enzymes with the particulate fraction of the red cell was assessed for six enzymes. No binding was detected for hexokinase, PK and LDH; low levels of binding by phosphofructokinase (5%), aldolase (1%) and glyceraldehyde-3-phosphate dehydrogenase (3%) were not altered by strenuous exercise. Glycolytic enzyme binding, therefore, does not appear to be an important regulator of energy metabolism in these cells. Intracellular glucose levels, in contrast, appeared to be regulated following exercise stress. An increase in intra-erythrocytic lactate levels at this time may reflect the importance of exogenously produced lactate as a substrate for ATP production. The description of trout red cell metabolism presented here provides a basis for further study of the relationships between organismic gas exchange and molecular-level adaptation of nucleated red cell function.

Glycolytic Enzyme Binding and Metabolic Control in Estivation and Anoxia in the LandSnail Otala Lactea

ABSTRACTThe mechanisms controlling glycolytic rate were examined in foot muscle of the terrestrial snail Otala lactea (Müller) (Pulmonata, Helicidae), during short and long periods of estivation and anoxia. Binding associations between glycolytic enzymes and the particulate fraction of the cell were assessed in both states. The percentage of enzyme activity bound to particulate matter decreased significantly over the short term (4 days estivation and 14·5 h anoxia); significant changes were seen for hexokinase (HK), phosphofructokinase (PFK), aldolase and lactate dehydrogenase (LDH) in estivation and, for these enzymes plus triosephosphate isomerase and pyruvate kinase (PK), in anoxia. Over the longer term in estivation (22 days) and anoxia (45 h), enzyme binding returned to control values. Tissue content of fructose-2,6-bisphosphate, a potent phosphofructokinase activator, decreased under all experimental conditions. Total glycogen phosphorylase activity decreased during short-term anoxia (14·5 h) and during long-term estivation (22 days), but the percentage of the active a form decreased significantly during anoxia only. Significant changes in the maximal activities of several enzymes were observed during both estivation and anoxia. Decreases in the maximal activity of HK, PFK, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (PGK) and LDH were observed during long-term estivation. Increases in PGK and PK maximal activity in short-term anoxia and aldolase and PGK in long-term anoxia were also observed. These results suggest that changes in glycolytic enzyme binding may be part of an immediate mechanism used to cause a rapid decrease in glycolytic flux and initiate glycolytic rate depression, which also includes a reduction of fructose-2,6-bisphosphate content and decreased glycogen phosphorylase activity. In the long term, however, control of snail glycolytic rate is reorganized, so that enzyme binding associations revert to the control values. In the long term, then, control is mediated by lower fructose-2,6-bisphosphate concentrations and, during estivation, also by a decrease in maximal enzyme activities.

Protein Assembly and Metabolic Regulation: Physiological and Evolutionary Perspectives

Many physiologically important adjustments of metabolic activities are achieved by modifying the assembly states of proteins. These assembly-state changes involve the reversible aggregation of the subunits of multi-subunit proteins and the reversible binding of enzymes to other enzymes or to structural components of cells (compartmentation). Adaptive shifts in protein assembly states are key elements in both rapid metabolic adjustments, such as those occurring in transitions from slow to rapid muscular activity, and longer-term metabolic changes, such as those associated with different types of dormancy. Changes in subunit assembly state allow sharp "on-off" regulation of enzymes, as manifested in the regulation of phosphofructokinase (PFK) and trehalase activities by transitions in pH. Changes in the aggregation states of multiprotein complexes alter the proximity of the several enzymes composing apathway, thereby altering the eiciency of metabolite transfer among enzymes of the sequence. Aggregation also may facilitate interaction between enzymes that generate a metabolite, for example, ATP, with other enzymes using this metabolite. The binding of glycolytic enzymes to the myofibrillar apparatus is a case in point. The enhancement of metabolic regulatory mechanisms made possible by these reversible changes in protein assembly is likely the selective advantage fostering the evolution of multiple protein-binding domains in many eukaryotic enzymes. A deeper understanding of multiprotein complexes, then, will not only advance knowledge of metabolic regulation, but in addition will help explain the evolutionary development oflarge, complex, and often highly conserved protein structures From an experimental standpoint, the differences in catalytic performance observed between a purified enzyme studied in a simple reaction mixture versus the same enzyme analyzed in a milieu that includes the otherproteins with which it interacts, and the modulators that regulate these protein-protein interactions, can be dramatic. This observation highlights the need for conducting enzymatic studies under conditions that simulate as closely aspossible the intracellular state.

The effect of fatigue on the binding of glycolytic enzymes in the isolated gastrocnemius of Rana pipiens

Fatigue of isolated gastrocnemius muscles from R. pipiens leads to a marked increase in the proportion of phosphofructokinase bound to the particulate fraction and a decrease in the binding of lactate dehydrogenase, pyruvate kinase, creatine phosphokinase and glyceraldehyde-3-phosphate dehydrogenase. Only the proportion of aldolase bound to the particulate fraction was unaffected by fatigue. This pattern was unchanged when fatigued muscles were extracted at pH 6.5 rather than 7.5. Thus, muscle fatigue leads to opposite changes in the binding of the glycolytic enzymes.

Evidence for glycolytic enzyme binding during anaerobiosis of the foot muscle ofPatella caerulea (L.)

The effect of anaerobiosis and aerobic recovery on the degree of binding of glycolytic enzymes to the particulate fraction of the cell was studied in the foot muscle of the marine molluscP. caerulea, in order to assess the role of glycolytic enzyme binding in the metabolic transition between aerobic and anoxic states. Short periods of anoxia (2 h, 4 h) resulted in an increase in enzyme binding in association with the increased glycolytic rate observed; this was particularly pronounced for phosphorylase, phosphofructokinase, aldolase, pyruvate kinase and lactate dehydrogenase. Decreased enzyme binding was observed after prolonged periods of anoxia. These effects were reversed and control values re-established when animals were returned to aerobic conditions. The results suggest that glycolytic rate could be regulated by changes in the distribution of glycolytic enzymes between free and bound forms inP. caerulea foot muscle. This reversible interaction of glycolytic enzymes with structural proteins may constitute an additional mechanism for metabolic control.

Interaction of fish muscle glycolytic enzymes with F-actin and actomyosin.

The binding of fish muscle glycolytic enzymes with F-actin and actomyosin was studied in vitro under various conditions. The binding of glucosephosphate isomerase, phosphoglycerate kinase and enolase with the myofibrillar proteins was weak, while that of aldolase and glyceraldehyde-3-phosphate dehydrogenase was strong. The latter enzymes showed the ratio of the unbound to total enzyme ranging from 9-50%, when mixed with F-actin or actomyosin. The recovery of bound enzyme ranged from 61 to 86% for aldolase and from 60 to 92% for glyceraldehyde-3-phosphate dehydrogenase.The enzyme-myofibrillar protein binding was depressed by the increase in reaction pH, KCl or Ca2+ concentration. In the presence of 150mM KCl or 50mM Ca2+, the binding was inhibited almost completely. Glycolytic metabolites also influenced the binding. Fructose-l, 6-bisphosphate and 2, 3-bisphosphoglycerate caused a marked adsorption-hindrance for aldolase at a concentration of 1mM. The results obtained could account for the enzyme-specific extractability from fish muscle.

The possible role of glycolytic enzyme binding in the control of glycolysis in Patella caerulea foot muscle during stimulation

1. 1. The extent of glycolytic enzyme activity associated with the particulate fraction of homogenates was measured in P. caerulea foot muscle at rest and after electrical stimulation. 2. 2. In association with the increased glycolytic rate on stimulation, there was a significant increase in the binding of certain enzymes such as phosphorylase, phosphoglucomutase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase. 3. 3. It is concluded that the variable enzyme binding to the particulate fraction could constitute an additional mechanism for metabolic control in P. caerulea foot muscle.

Reevaluation of the “glycolytic complex” in muscle: A multitechnique approach using trout white muscle

Preliminary characterization of the “glycolytic complex,” formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a p K a of 7.3 in 30 m m imidazole. PFK binding increased at lower pH values; at 150 m m KC1 the apparent p K a value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium—with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F. D. Shaw, and D. J. Morton (1980) Biochem. J. 186, 105–109) . In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10–30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.

Subcellular enzyme binding in glycolytic control: in vivo studies with fish muscle.

The effect of exercise on the binding of glycolytic enzymes to subcellular structures was examined in rainbow trout (Salmo gardneri). Both "burst" and "endurance" type exercise produced an increase of approximately 50% in the percentage of phosphofructokinase (PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase, and 3-phosphoglycerate kinase associated with particulate matter in white skeletal muscle. In contrast, cardiac muscle showed no change in enzyme binding when trout were exercised, suggesting that the effects seen in white muscle are related to an increased anaerobic glycolytic flux in this tissue. Modulation of binding by altered pH or calcium ion concentration was tested: a decrease in pH increased PFK binding to subcellular particles, whereas 2 mM CaCl2 decreased GAPDH binding. These results are discussed with respect to the formation of a glycolytic complex during exercise in trout white muscle.

Binding of Glycolytic Enzymes to a Particulate Fraction in Carrot and Sugar Beet Storage Roots

Numerous studies have demonstrated a rapid increase in the respiration rate during aging of slices of tuber and storage roots. To determine the molecular mechanisms of this phenomenon, the role of enzyme binding to the subcellular particulate fraction has been assessed in carrot (Daucus carota L.) and sugar beet (Beta vulgaris L.). Soluble versus particulate fractions were separated by centrifugation at 16,000g and both fractions assayed for the activities of six glycolytic enzymes. Preparations from sliced and aged tissues showed elevated percentages of five enzymes associated with the particulate fraction as compared with controls. The stimulation of respiration which occurs during aging of underground storage organ slices may result, in part, from an association of enzymes with the particulate fraction of the cell promoting an elevated glycolytic rate.