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.     

The influence of insulin and glucagon on the interactions between glycolytic enzymes and cellular structure

The influence of insulin and glucagon on the release of glycolytic enzyme activities and actin from cultured pig kidney cells treated with digitonin has been studied. Both insulin and glucagon reduced the release of all glycolytic enzymes except for phosphofructokinase, and concurrently reduced the release of actin. These data have been discussed in relation to their contribution to knowledge of the interactions between glycolytic enzymes and actin filaments of the cytoskeleton, and to the influence of hormones on these interactions.     

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.     

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.Abbreviation pH_i intracellular concentration of H⁺ ions     

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 electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications.

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.     

Comparative levels between enzymes of the glycolytic pathway from erythrocytes and somatic tissues of the chicken Gallus gallus domesticus

A comparative study on the glycolytic enzymes from chicken erythrocytes and somatic tissues has been carried out, the results being shown as active units per mg protein in supernatants of 1085, 12,100 and 106,000 g fractionated centrifugation. The profiles of the glycolytic enzymes have been analyzed in terms of their activity relative to hexokinase and as the ratios between pairs of enzymes bearing a product-substrate relationship. Chicken erythrocyte displays a very peculiar profile of glycolytic enzymes. It possesses a FruP2-activated pyruvate kinase of the L isoenzyme type, which does not seem to be the predominant isoenzyme together with the M type, the content in glycolytic enzymes being much lower than in the somatic tissues.     

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.     

Haemonchus contortus: The in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes

Kaur R. and Sood M. L. 1982. Haemonchus contortus: the in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes. International Journal for Parasitology 12: 585–588. Various enzymes of glycolysis (hexokinase, phosphoglucomutase, phosphoglucoisomerase, adolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase-enolase-pyruvate kinase and lactate dehydrogenase) have been detected in adult Haemonchus contortus. Low pyruvate kinase and lactate dehydrogenase activities suggested an alternate pathway from phosphoenolpyruvate. In vitro incubation had no significant effects on these enzymes and the worm was able to maintain normal metabolism for 12 h. Varying degrees of inhibition of glycolytic enzymes were observed with 50 μg/ml of dl-tetramisole and rafoxanide. The enzymes were inhibited to a greater extent by dl-tetramisole. These effects may block the glycolytic pathway and deprive the parasite of its ATP production.     

Relative phosphate contents of phosphofructokinase and other soluble phosphoproteins in skeletal muscle

In vivo phosphorylation of muscle proteins has been studied by incorporation of [³²P]phosphate with emphasis placed upon the phosphorylation of glycolytic enzymes. Of the approximately 25 soluble proteins resolved by two-dimensional electrophoresis that contain significant ³²P, phosphofructokinase was the sole glycolytic enzyme identified as a phosphoprotein. The extent of phosphorylation found for this enzyme was the same as determined previously for purified phosphofructokinase and was about the same as the extent of phosphorylation of phosphorylase in resting muscle. Subsequent partial purification of several glycolytic enzymes confirmed the absence of significant amount of phosphate. However, phosphoglycerate mutase contained small amounts of covalently bound ³²P that was exchangeable with 3-phosphoglycerate and therefore, most likely was incorporated during the catalytic reaction cycle. Analogous results were obtained for phosphoglucomutase. Both mutases were also phosphorylated at the same sites by the catalytic subunit of cyclic AMP-dependent protein kinase.     

Haemonchus contortus: The in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes

Kaur R. and Sood M. L. 1982. Haemonchus contortus: the in vitro effects of dl-tetramisole and rafoxanide on glycolytic enzymes. International Journal for Parasitology 12: 585–588. Various enzymes of glycolysis (hexokinase, phosphoglucomutase, phosphoglucoisomerase, adolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase-enolase-pyruvate kinase and lactate dehydrogenase) have been detected in adult Haemonchus contortus. Low pyruvate kinase and lactate dehydrogenase activities suggested an alternate pathway from phosphoenolpyruvate. In vitro incubation had no significant effects on these enzymes and the worm was able to maintain normal metabolism for 12 h. Varying degrees of inhibition of glycolytic enzymes were observed with 50 μg/ml of dl-tetramisole and rafoxanide. The enzymes were inhibited to a greater extent by dl-tetramisole. These effects may block the glycolytic pathway and deprive the parasite of its ATP production.     

Association of C-terminal region of phosphoglycerate mutase with glycolytic complex regulates energy production in cancer cells

Cancer cells prefer anaerobic ATP synthesis, regardless of the availability of oxygen. It has been hypothesized that in these cells, glycolytic enzymes associate into a large complex, which results in an increased efficiency of glycolytic flux. However, there is no convincing in vivo evidence supporting this hypothesis. Here, we show that all the enzymes of triose phosphate metabolism, from aldolase to pyruvate kinase consecutively, form a macromolecular complex in vivo and that disruption of such complex significantly inhibits lactate release and ATP synthesis in the glycolytic pathway. Composition of the complex and the effectiveness of the glycolytic flux depends on lactate and glucose concentration. High concentrations of exogenous lactate reduces association of the C-terminal region phosphoglycerate mutase (PGAM) with the complex which results in its disruption and inhibition of ATP synthesis. Additionally, high lactate affects nuclear localization of PGAM and ceases cell proliferation. Our findings might provide new prospects for cancer treatment using low-molecular weight competitors to destabilize the glycolytic complex and reduce proliferative potential of cancer cells.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

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.     

How to keep glycolytic metabolite concentrations constant when ATP/ADP and NADH/NAD+ change

Glycolytic flux may increase over 100 times in skeletal muscle during rest-to-work transition, whereas glycolytic metabolite concentrations remain relatively constant. This constancy cannot be explained by an identical direct activation of all glycolytic enzymes because the concentrations of ATP, ADP, AMP, P(i), NADH and NAD+, modulators of the activity of different glycolytic enzymes, change. It is demonstrated in the present in silico study that a perfect homeostasis of glycolytic metabolite concentrations can be achieved if glycolysis is divided into appropriate blocks of enzymes that are directly activated to a different extent in order to compensate the effect of the modulators.     

Evolving concepts in plant glycolysis: two centuries of progress

Glycolysis, the process responsible for the conversion of monosaccharides to pyruvic acid, is a ubiquitous feature of cellular metabolism and was the first major biochemical pathway to be well characterized. Although the majority of glycolytic enzymes are common to all organisms, the past quarter of a century has revealed that glycolysis in higher plants possesses numerous distinctive features. Research in the nineteenth century established convincingly that plants carry out alcoholic fermentation under anaerobic conditions. In 1878, Wilhelm Pfeffer asserted that a non-oxygen-requiring ‘intramolecular respiration’ was involved in the aerobic respiration of plants. Between 1900 and 1950 it was demonstrated that plants metabolize sugar and starch by a glycolytic pathway broadly similar to that of yeasts and muscle tissue. In 1948, the first purification and characterization of a plant glycolytic enzyme, aldolase, was published by Paul Stumpf. By 1960 the presence of each of the 10 enzymes of glycolysis, presumed at the time to be located in the cytosol, had been confirmed in higher plants. Shortly after 1960 it was shown that the mechanism of glycolytic regulation in plants had features in common with that of animals and yeasts, especially as regards the important role played by the enzyme phosphofructokinase; but important regulatory properties peculiar to plants were soon demonstrated. In the last 30 years, higher-plant glycolysis has been found to exhibit a number of additional characteristics peculiar to plant systems. One conspicuous feature of plant glycolysis, discovered in the 1970s, is the presence of a complete or nearly complete sequence of glycolytic enzymes in plastids, distinct and spatially separated from the glycolytic enzymes located in the cytosol. Plastidic and cytosolic isoenzymes of glycolysis have been shown to differ in their kinetic and regulatory properties, suggesting that the two pathways are independently regulated. Since about 1980 it has become increasingly clear that the cytosolic glycolysis of plants may make use of several enzymes other than the conventional ones found in yeasts, muscle tissue and plant plastids: these enzymes include a pyrophosphate-dependent phosphofructokinase, a non-reversible and nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, a phosphoenolpyruvate phosphatase (vacuolar location) and a three-enzyme sequence able to produce pyruvate from phosphoenolpyruvate avoiding the pyruvate-kinase step. These non-conventional enzymes may catalyze glycolysis in the plant cytosol especially under conditions of metabolic stress. Experiments on transgenic plants possessing significantly elevated or reduced (reduced to virtually nil in some cases) levels of glycolytic enzymes are currently playing an important part in improving our understanding of the regulation of plant glycolysis; such experiments illustrate an impressive degree of flexibility in the pathway's operation. Plant cells are able to make use of enzymes bypassing or substituting for several of the conventional enzymic steps in the glycolytic pathway; the extent and conditions under which these bypasses operate are the subject of current research. The duplication of the glycolytic pathway in plants and the flexible nature of the pathway have possibly evolved in relation to the crucial biosynthetic role played by plant glycolysis beyond its function in energy generation; both functions must proceed if a plant is to survive under varying and often stressful environmental or nutritional conditions.     

Studies on glycolysis in vitro: role of glucose phosphorylation and phosphofructokinase activity on total velocity

An in vitro glycolysis system has been developed to study the regulation of glycolysis on kinetic structure basis, in order to determine the extent of regulatory effects on the whole system of individual enzymes according to their kinetic data, in rat liver and muscle. Hexokinase or glucose-6-phosphate addition to the system with glucose as substrate increases lactate production rate by 2.5 in liver and by 10 in muscle, which suggest glucose phosphorylation step is a limiting step in this system. Fructose 2,6-bisphosphate addition to the system increases lactate production rate in liver only when glucose is the substrate, but not with glucose-6-phosphate as substrate. There is a linear relationship between glycolytic activity, as lactate produced per min and protein quantity, which suggests that this system can also be used to assay glycolytic activity in tissue extracts. Specific glycolytic activity found, as mumol of L-lactate produced per min, per protein mg was 0.1 for muscle and 0.01 for liver.     

Yeast glycolytic oscillations that are not controlled by a single oscillophore: a new definition of oscillophore strength

Biochemical oscillations, such as glycolytic oscillations, are often believed to be caused by a single so-called ‘oscillophore’. The main characteristics of yeast glycolytic oscillations, such as frequency and amplitude, are however controlled by several enzymes. In this paper, we develop a method to quantify to which extent any enzyme determines the occurrence of oscillations. Principles extrapolated from metabolic control analysis are applied to calculate the control exerted by individual enzymes on the real and imaginary parts of the eigenvalues of the Jacobian matrix. We propose that the control exerted by an enzyme on the real part of the smallest eigenvalue, in terms of absolute value, quantifies to which extent that enzyme contributes to the emergence of instability. Likewise the control exerted by an enzyme on the imaginary part of complex eigenvalues may serve to quantify the extent to which that enzyme contributes to the tendency of the system to oscillate. The method was applied both to a core model and to a realistic model of yeast glycolytic oscillations. Both the control over stability and the control over oscillatory tendency were distributed among several enzymes, of which glucose transport, pyruvate decarboxylase and ATP utilization were the most important. The distributions of control were different for stability and oscillatory tendency, showing that control of instability does not imply control of oscillatory tendency nor vice versa. The control coefficients summed up to 1, suggesting the existence of a new summation theorem. These results constitute proof that glycolytic oscillations in yeast are not caused by a single oscillophore and provide a new, subtle, definition for the oscillophore strength of an enzyme.     

Comparative levels of muscle glycolytic enzymes in mammals, fish, echinoderm and molluscs.

1. Levels of glycolytic enzymes were determined in terms of units of enzyme/mg protein in rat striated muscle, carp lateral muscle, holothuria longitudinal muscle of the body wall, and a snail foot muscle. 2. An attempt has been made to correlate levels of glycolytic enzymes as a parameter to establish a "biochemical distance" at molecular level and correlate this with the phylogenetic position in animals sufficiently separated in the animal tree of evolution. 3. The possibility of a peculiar kinetic behaviour of the glycolytic pathway in each muscle tissue studied, has been analyzed as the profiles of the ratios of pairs of enzymes bearing a substrate-product dependence. 4. A possible "futile synthesis" of some glycolytic enzymes, such as FDP-aldolase in the case of fish muscle, is proposed.     

Regulation of skeletal muscle metabolism by enzyme binding.

The random diffusion mechanism is usually assumed in analyzing the energetics of specific pathways despite the findings that enzymes associate with each other and (or) with various membranous and contractile elements of the cell. Successive glycolytic enzymes have been shown to associate in the cytosol as enzyme complexes or bind to the thin filaments. Furthermore, the degree of glycolytic enzyme interactions have been shown to change with altered rates of carbon flux through the pathway. In particular, the proportions of aldolase, phosphofructokinase, and glyceraldehyde phosphate dehydrogenase bound to the contractile proteins have been found to increase with increased rates of glycolysis. In addition, decreasing pH and ionic strength are also associated with an increase in glycolytic enzyme interactions. The kinetics displayed by interacting enzymes generally serve to enhance their catalytic efficiencies. The associations of the glycolytic enzymes serve to enhance metabolite transfer rates, increase the local concentrations of intermediates, and provide for regulation of activity via effectors. Therefore these interactions provide an additional mechanism for regulating glycolytic flux in skeletal muscle.