The influence of deoxyribonuclease I and cytochalasin D on the release of glycolytic enzymes from digitonized cells

In permeabilized cells, deoxyribonuclease I has been demonstrated to cause a decrease in the extent of binding to cellular structure of all of the glycolytic enzymes other than phosphofructokinase, with this decrease being most marked for aldolase and glyceraldehydephosphate dehydrogenase. Cytochalasin D, in contrast, did not produce this type of effect. These results have been discussed in relation to the evidence for the existence of a complex of glycolytic enzymes which binds to elements of the cytoplasmic matrix, and the possible organization of this complex.     

Effect of phorbol myristate acetate and concanavalin A on the glycolytic enzymes of human peripheral lymphocytes

The effects of Concanavalin A and the tumor promoting agent, phorbol 12-myristate 13-acetate (PMA), on glycolytic enzymes in human peripheral lymphocytes have been studied. A combination of Concanavalin A plus PMA stimulates DNA and protein synthesis to a significantly greater extent than when each are added individually. PMA and concanavalin A together, but not individually, also increase the levels of the activity of the glycolytic enzymes in peripheral lymphocytes treated for 48 h. The increase in hexokinase activity induced by PMA plus concanavalin A appeared to be due to the expression of the isoenzyme form, hexokinase II. The results suggest that the expression of glycolytic enzymes in stimulated lymphocytes is a late event (perhaps associated with the S phase) which is regulated by a cellular signal system controlled by the combined action of PMA plus concanavalin A.     

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.     

Studies on associations of glycolytic and glutaminolytic enzymes in MCF-7 cells: Role of P36

Isoelectric focusing of MCF-7 cell extracts revealed an association of the glycolytic enzymes glyceraldehyde 3-phosphate-dehydrogenase, phosphoglycerate kinase, enolase, and pyruvate kinase. This complex between the glycolytic enzymes is sensitive to RNase. p36 could not be detected within this association of glycolytic enzymes; however an association of p36 with a specific form of malate dehydrogenase was found. In MCF-7 cells three forms of malate dehydrogenase can be detected by isoelectric focusing: the mitochondrial form with an isoelectric point between 8.9 and 9.5, the cytosolic form with pl 5.0, and a p36-associated form with pl 7.8. The mitochondrial form comprises the mature mitochondrial isoenzyme (pl 9.5) and its precursor form (pl 8.9). Refocusing of the pl 7.8 form of malate dehydrogenase also gave rise to the mitochondrial isoenzyme. Thus, the pl 7.8 form of malate dehydrogenase is actually the mitochondrial isoenzyme retained in the cytosol by the association with p36. Addition of fructose 1,6-bisphosphate to the initial focusing column induced a quantitative shift of the pl 7.8 form of malate dehydrogenase to the mitochondrial forms (pl 8.9 and 9.5). In MCF-7 cells p36 is not phosphorylated in tyrosine. Kinetic measurements revealed that the pl 7.8 form of malate dehydrogenase has the lowest affinity for NADH. Compared to both mitochondrial forms the cytosolic isoenzyme has a high capacity when measured in the NAD → NADH direction (malate → oxaloacetate direction). The association of p36 with the mitochondrial isoenzyme may favor the flow of hydrogen from the cytosol into the mitochondria. Inhibition of cell proliferation by AMP which leads to an inhibition of glycolysis has no effect on complex formation by glycolytic and glutaminolytic enzymes in MCF-7 cells. AMP treatment leads to an activation of malate dehydrogenase, which correlates with the increase of pyruvate and the decrease of lactate levels, but has no effect on the distribution of the various malate dehydrogenase forms. © 1996 Wiley-Liss, Inc.     

Distribution of isoenzymes of the glycogenolytic cascade in different types of muscle fibre.

Distinct isoenzyme patterns of the glycogenolytic enzymes exist in different fibre types. Fast twitch glycolytic and slow twitch oxidative fibres differ in the proportion of the two isoenzymes of cyclic AMP dependent protein kinase and in the type of phosphorylase kinase that is present. Slow twitch oxidative fibres and cardiac fibres resemble one another in these two respects, but differ in that the type I phosphorylase of cardiac muscle is absent in slow twitch oxidative fibres. In all examples, the functional differences between the isoenzymes seem to be related to the regulatory rather than the catalytic behavior of the molecules. In the case of cyclic AMP dependent protein kinase and phosphorylase kinase, it is a regulatory subunit that appears to be affected [16,23], while in the case of phosphorylase, the type I isoenzyme is known to have a five to eight-fold Ka for the allosteric activator 5' AMP [6]. However, the precise physiological significance of these differences remains to be elucidated.     

Isoelectric forms of alpha-L-fucosidase in mouse teratocarcinoma-derived cell lines

The alpha-L-fucosidase isoenzyme pattern of mouse teratocarcinoma-derived cell lines was analyzed by isoelectric focusing and compared with the pattern of a mammary carcinoma as an example of a malignant somatic cell line. In addition, these isoenzyme patterns were compared with those of normal fetal and adult mouse tissues from an earlier study. In the normal early fetal and placental tissues as well as in embryonal carcinoma and yolk sac carcinoma cells the alpha-L-fucosidase activity is predominantly associated with basic forms of the enzyme. This embryonic pattern of alpha-L-fucosidase is characterized by one to three isoelectric forms of the enzyme with pI values ranging from 7 to 9.5 accounting for more than two-thirds of the total activity. In contrast, the mammary carcinoma pattern resembles adult somatic tissues and primarily expresses acidic enzymatic forms (which comprise approximately 80% of total activity). The somatic cell malignancies arising in retransplantable teratocarcinomas show varying isoenzyme patterns. Thus, a malignant fibrous histiocytoma expresses predominantly basic forms of the enzyme, whereas a leiomyosarcoma expresses approximately equal amounts of acidic and basic forms of the enzyme resembling in this respect late fetal or immature neonatal tissues. These findings show that the embryonal carcinoma and yolk sac carcinoma cells of the mouse express the embryonic isoenzyme pattern of alpha-L-fucosidase in contrast to malignant cells originating in somatic tissue, like mammary carcinoma, which express the adult pattern. Malignancies arising in somatic tissues of teratocarcinomas may retain the embryonic alpha-L-fucosidase phenotype or show a phenotype corresponding to late fetal or neonatal tissues in normal ontogeny.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Different internal metabolites trigger the induction of glycolytic gene expression in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the sugar-induced expression of various genes coding for glycolytic enzymes is triggered by increases in the concentrations of different internal metabolites. Here, we show that the induction of the glycolytic isoenzyme enolase 2 is strictly dependent on the abilities of different mutant strains to increase the level of glucose-6-phosphate after the addition of sugars. In contrast, the induction of alcohol dehydrogenase I is dependent on increasing concentrations of metabolites in the late stages of glycolysis.     

On the differential release of glycolytic enzymes from cellular structure

In an endeavour to extend the available information on the biological significance of the interactions between glycolytic enzymes and cellular ultrastructure, the role of release of enzymes from digitonized fibroblasts has been studied. Lactate dehydrogenase and phosphofructokinase were rapidly and quantitatively eluted under the experimental conditions, while glyceraldehyde-3-phosphate dehydrogenase and aldolase were retained to an appreciably greater extent by the cells. This differential release of glycolytic enzymes has been related to the known binding propensities between those enzymes and subcellular structures, and are interpreted as providing additional confirmatory evidence of the importance of aldolase and glyceraldehyde-3-phosphate dehydrogenase, in particular, to these associations. The data also shed light on the order of binding of these glycolytic components - phosphofructokinase being indicated as binding subsequently (and probably separately) to aldolase and glyceraldehyde-3-phosphate dehydrogenase. These results have been discussed in relation to the available data on the associations between glycolytic enzymes and cellular structure, the possible physiological significance of this phenomenon, and the access to these problems provided by the present technique.     

Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming

The bioenergetics of somatic dedifferentiation into induced pluripotent stem cells remains largely unknown. Here, stemness factor-mediated nuclear reprogramming reverted mitochondrial networks into cristae-poor structures. Metabolomic footprinting and fingerprinting distinguished derived pluripotent progeny from parental fibroblasts according to elevated glucose utilization and production of glycolytic end products. Temporal sampling demonstrated glycolytic gene potentiation prior to induction of pluripotent markers. Functional metamorphosis of somatic oxidative phosphorylation into acquired pluripotent glycolytic metabolism conformed to an embryonic-like archetype. Stimulation of glycolysis promoted, while blockade of glycolytic enzyme activity blunted, reprogramming efficiency. Metaboproteomics resolved upregulated glycolytic enzymes and downregulated electron transport chain complex I subunits underlying cell fate determination. Thus, the energetic infrastructure of somatic cells transitions into a required glycolytic metabotype to fuel induction of pluripotency.     

On the existence of a complex of glycolytic enzymes

It has been suggested in the literature that the glycolytic enzymes are organized into a multi-enzymic complex. We have evaluated this hypothesis for the phosphotriose-glycerate phosphate group of glycolytic enzymes of muscle using sucrose density gradient centrifugation, gel filtration, and ultrafiltration. Attempts were made to avoid dilution and changes in pH. The ratio of activities of the phosphotriose-glycerate phosphate group of enzymes was similar to that found in several other tissues that has led to their designation as a constant proportion group of enzymes. However, no evidence was obtained that they exist as a multi-enzymic complex in chicken breast muscle. As the pH of the press juice is raised to 7.0 and the temperature to 25°C, association occurs between some components in the muscle press juice as evidenced by a blocking of the pores of an ultrafiltration membrane. This association, however, does not involve the enzymes of the phosphotriose-glycerate phosphate group.     

Trigonella foenum graecum (fenugreek) seed powder improves glucose homeostasis in alloxan diabetic rat tissues by reversing the altered glycolytic,gluconeogenic and lipogenic enzymes

Trigonella foenum graecum (fenugreek) seed powder has been suggested to have potential antidiabetic effects. The effect of oral administration of Trigonella whole seed powder (5% in the diet) for 21 days on glycolytic, gluconeogenic and NADPlinked lipogenic enzymes were studied in liver and kidney tissues of alloxan-induced diabetic Wistar rats. Diabetic rats were characterised by a 4fold higher blood glucose level and a 0.7fold lower body weight compared to normal controls. The activities of the glycolytic enzymes were significantly lower in the diabetic liver and higher in the diabetic kidney. The activities of gluconeogenic enzymes were higher in both liver and kidney during diabetes, however the activities of the lipogenic enzymes were decreased in both tissues during diabetes. Trigonella seed powder treatment to diabetic rats for 21 days brought down the elevated fasting blood glucose levels to control levels. The altered enzyme activities were significantly restored to control values in both the liver and kidney after Trigonella seed powder treatment. The therapeutic role of Trigonella seed powder in type1 diabetes as exemplified in this study can be attributed to the change of glucose and lipid metabolising enzyme activities to normal values, thus stabilizing glucose homeostasis in the liver and kidney. These biochemical effects exerted by Trigonella seeds make it a possible new therapeutic in type1 diabetes.     

Ontogenetic Development of Creatine Phosphokinase in Skeletal Muscles and Heart from Pigs

Creatine Phosphokinase (CPK) in striated muscles shows only small changes in activity before birth. After birth and during the first month of extrauterine life the activity increases rapidly. The largest increase is seen in muscles with a glycolytic energy metabolism (m. long, dorsi) and the smallest in muscles with an oxydative energy metabolism (m. flexor dig. ped. sup.). The differences between these groups of muscles are statistically significant. In heart tissue the increase in CPK activity is lower, the levels amounting to 40 to 47 % of those in striated muscles. Early in fetal life only the BB isoenzyme is found in striated muscles. Synthesis of M subunits of GPK starts between day 76 and 65 before birth and increases rapidly after this time leading to disappearance of the BB isoenzyme 24 days prior to birth and of the MB isoenzyme at birth. In muscles with an oxydative as well as in muscles with a glycolytic metabolism all GPK activity after birth is caused by the MM isoenzyme. All three isoenzymes are present in heart tissue at the earliest prenatal stage investigated, the pattern being dominated by the BB isoenzyme. During further differentiation the MM isoenzyme increases and the BB isoenzyme decreases. The development is completed during the first month after birth with a final isoenzyme composition of 81 % MM and 19 % MB isoenzyme. kw|Keywords|k]pigs; k]ontogenesis; k]creatine phosphokinase; k]activity; k]isoenzymes     

Metabolic implications of the distribution of the alanine aminotransferase isoenzymes.

The distribution of alanine aminotransferase isozymes in several tissues from several species has been studied. In glycolytic tissues, such as skeletal and cardiac muscle, cytosolic alanine aminotransferase was the predominant form. In gluconeogenic tissues, such as liver and kidney, the concentration of the cytosolic alanine aminotransferase was much more variable; its presence, however, may be correlated with the presence of phosphoenolpyruvate carboxykinase in the same compartment. The particulate enzyme was found associated only with the matrix of the mitochondria. It was present only in those gluconeogenic tissues that can utilize alanine for glucose production, e.g. rat liver and pig liver and kidney; it was absent from rat kidney which cannot convert alanine to glucose. These observations, together with the kinetic parameters of the two isozymes, suggest that in vivo, mitochondrial alanine aminotransferase is involved in the conversion of alanine to pyruvate, while the cytosolic isoenzyme is mainly involved in the formation of alanine from pyruvate.     

Human glutathione S-transferases: radioimmunoassay studies on the expression of alpha-, mu- and pi-class isoenzymes in developing lung and kidney

The developmental expression of the alpha-, mu- and pi-class glutathione S-transferases has been defined in human lung and kidney using radioimmunoassay, immunohistochemistry and column chromatography. Expression of alpha-class enzymes increased significantly after about 40 weeks gestation in kidney but not lung, while expression of mu isoenzymes was continuous throughout development in both tissues. Expression of the pi isoenzyme fell during in utero ontogeny in lung, the pattern of down-regulation being similar to that previously observed in liver. There was no change in the expression of this isoenzyme in kidney. Comparison of the expression of the glutathione S-transferases in developing lung, kidney and liver shows some common patterns of expression suggesting these genes are under similar regulatory control.     

Glycolytic adjustments in tissues of frog Rana ridibunda and land snail Helix lucorum during seasonal hibernation

The present work aimed to contribute to the understanding of the adaptation of the glycolytic pathway in tissues of frog Rana ridibunda and land snail species Helix lucorum during seasonal hibernation. Moreover responses of glycolytic enzymes from cold acclimated R. ridibunda and H. lucorum were studied as well. The drop in Po₂ in the blood of hibernated frogs and land snails indicated lower oxygen consumption and a decrease in their metabolic rate. The activities of glycolytic enzymes indicated that hibernation had a differential effect on the glycolyis in the two species studied and also in the tissues of the same species. The activity of l-LDH decreased significantly in the skeletal muscle and heart of hibernated R. ridibunda indicating a low glycolytic potential. Similar biochemical responses were observed in the same tissues during cold acclimation. The continuous increase in the activities of glycolytic enzymes studied, except for HK, might indicate a compensation for the impacts of low temperature on the enzymatic activities. In contrast to R. ridibunda, the activities of the enzymes increased and remained at higher levels than those of the prehibernation controls indicating maintenance of glycolytic potential in the tissues of hibernating land snails.     

Content and synthesis of glycolytic enzymes and creatine kinase in skeletal muscles and normal and dystrophic chickens

A number of workers have reported that avian muscular dystrophy causes alterations in the levels of certain enzyme activities in "fast-twitch" muscle fibers but has little effect on enzyme activities in "slow-twitch" muscle fibers. In the present work, the effects of this disease on the content and relative rates of synthesis of a number of glycolytic enzymes and the skeletal muscle-specific MM isoenzyme of creatine kinase in chicken muscles was investigated. It was shown that (i) the approximate 50% reductions in steady-state concentrations of three glycolytic enzymes (aldolase, enolase, and glyceraldehyde-3-P dehydrogenase) in dystrophic breast (fast-twitch) muscle result predominantly from decreases in relative rates of synthesis, rather than accelerations in relative rates of degradation, of these proteins in the diseased tissue; (ii) in contrast to the situation with the glycolytic enzymes, muscular dystrophy has only minor effects (25% or less) on the content and relative rate of synthesis of MM creatine kinase in breast muscle fibers; (iii) the muscular dystrophy-associated alterations in content and synthesis of the glycolytic enzymes in breast muscle fibers become apparent only during postembryonic maturation of this tissue; and (iv) as expected, muscular dystrophy has no significant effect on the content or relative rates of synthesis of glycolytic enzymes in slow-twitch lateral adductor muscles of the chicken. These results are discussed in terms of the apparent similarities between the effects of muscular dystrophy and surgical denervation on the protein synthetic programs expressed by mature fast-twitch muscle fibers.     

The influence of fructose-1:6-bisphosphate on the release of glycolytic enzymes from cellular structure

In order to provide information on the relative binding characteristics of glycolytic enzymes, the effect of fructose-1,6-bisphosphate (FBP) on the release of glycolytic enzymes from cultured pig kidney cells treated with digitonin has been studied. In the absence of FBP, a differential release of these enzymes was observed, with the order of retention being aldolase greater than glyceraldehyde-3-phosphate dehydrogenase greater than glucosephosphate isomerase, triosephosphate isomerase, phosphoglycerokinase, phosphoglucomutase, lactate dehydrogenase, enolase, pyruvate kinase and phosphofructokinase. In the presence of fructose-1,6-bisphosphate, the release of aldolase was considerably enhanced, whereas the release of phosphofructokinase and pyruvate kinase was decreased by this metabolite. No significant alterations in the rate of release of the other enzymes was caused by FBP. These data have been discussed in relation to their contribution to the knowledge of the degree of association and order of binding between glycolytic enzymes and the cytoplasmic matrix.     

The influence of calcium ions on the adsorption of glycolytic enzymes to cellular structure

In order to provide information on the influence of Ca2+ ions on the adsorption of glycolytic enzymes to cellular structure, the release of these enzymes from digitonized cells has been studied. Increases in the calcium ion concentration were found to cause corresponding decreases in the extent of release of all the glycolytic enzymes, as well as a parallel increase in the extent of polymerization of actin. These observations have been discussed in relation to the effect of physiological concentrations of these ions on the association between glycolytic enzymes and the cytoskeleton.