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.     

Enzymic differentiation in pathways of carbohydrate metabolism in developing brain

The entire sequence of enzymes of the pentose phosphate pathway, the glycolytic pathway and certain enzymes of fatty acid synthesis were measured in developing rat brain. Coordinated increases in enzymes of the glycolytic pathway were observed, with apparent discrimination in that greater changes were found among enzymes of low turnover capability (Vmax: K_m). During development a sequential pattern emerged with early changes in phosphofructokinase and aldolase followed by triosephosphate isomerase, glycerophosphate dehydrogenase and phosphoglyceromutase; enzymes of the pentose phosphate pathway remained relatively constant, while a marked and parallel fall was seen in acetyl CoA car?ylase, fatty acid synthetase and NADP⁺-linked isocitrate dehydrogenase.     

Carbohydrate metabolism in Brugia pahangi (Nematoda: Filarioidea)

Carbohydrate metabolism in Brugia pahangi (Nematoda:Filarioidea). International Journal for Parasitology16: 465–469. Adult B. pahangi have a complete glycolytic sequence and tricarboxylic acid cycle. The activities of the glycolytic enzymes are extremely high, but their relative activities are similar to those found in other glycolytic tissues. A comparison of the mass action ratios of the glycolytic enzymes with their apparent equilibrium constants indicates that phosphorylase, hexokinase, phosphofructokinase and pyruvate kinase are non-equilibrium regulatory enzymes. There is also some evidence that aldolase may be pseudoregulatory. Adult B. pahangi have measurable amounts of fumarate reductase as well as phosphoenolpyruvate carboxykinase and NADP-linked malic enzyme. Apart from lactate. the only other acidic end-product detected was traces of alanine; lactate production was little affected by the presence or absence of oxygen, unless exogenous glucose was present.     

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.     

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.     

Hypothetical structure of the glycolytic enzyme complex (glycolytic metabolon) formed on erythrocyte membranes

A hypothetical structure of the glycolytic enzyme complex (glycolytic metabolon) adsorbed on the inner surface of the erythrocyte membrane has been proposed. Oligomers of integral membrane protein, band 3 protein (anion-transport system), are the anchor site for the complex. The complex is supposed to have a three-fold symmetry axis, perpendicular to the membrane plane, and contains a triple set of the glycolytic enzymes. The complex is in equilibrium with free enzymes; the equilibrium state depends on the physiological state of the erythrocyte.     

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     

Studies on cell-free metabolism: Ethanol production by extracts of Zymomonas mobilis

Using a cell-free extract of Zymomonas mobilis, it has been possible to achieve rapid and sustained ethanol production from added glucose. In one example 18% glucose was totally converted to 9% (w/v) ethanol. The controls on the glycolytic enzymes have been investigated by measuring metabolite levels during the experiment. No substantial accumulations of intermediates occurred when ATP production by the glycolytic metabolism was correctly balanced by ATPase activity. But as alcohol levels increased, some inhibitions of glucose 6-phosphate and pyruvate removal became apparent.     

Biochemical characterization and crystallization of recombinant 3-phosphoglycerate kinase of Plasmodium falciparum

Human malaria parasite Plasmodium falciparum depends largely on glycolytic pathway for energy metabolism during the intraerythrocytic life stage. Therefore, enzymes of the glycolytic pathway could offer potential drug targets provided novel biochemical and/or structural features of the parasitic enzymes, which distinguish them from the host counterpart, could be identified. 3-Phosphoglycerate kinase (EC 2.7.2.3) catalyzes an important phosphorylation step leading to the production of ATP in the glycolytic pathway. We have expressed recombinant 3-phosphoglycerate kinase of P. falciparum in Escherichia coli. The recombinant protein purified from the soluble fraction of E. coli is enzymatically active. The apparent K(m) values determined for adenosine triphosphate (ATP) and 3-phosphoglycerate (3-PGA) are 0.63 and 0.52 mM, respectively. The enzyme activity was temperature-sensitive. Suramin was found to inhibit the recombinant enzyme with an IC(50) value of 7 microM. We have crystallized the enzyme form in hexagonal space group P6(1)22 (or its enantiomorphic space group) with unit cell parameters a=b=130.7, c=263.9 A. Native data have been collected at 3.0-A resolution.     

48株爱德华氏菌糖酵解持家酶的胞外分泌

【背景】病原菌的糖酵解持家酶能分泌到胞外或定位在细胞膜表面,在病原菌的侵染和细胞粘附方面发挥着重要作用,爱德华氏菌是重要的鱼类致病菌,研究其糖酵解持家酶的胞外分泌有助于该病原的致病机制研究和疫苗开发。【目的】探究爱德华氏菌中糖酵解持家酶的胞外分泌。【方法】通过ELISA方法考察48种不同来源的爱德华氏菌中5种糖酵持家酶的胞外分泌。【结果】48种不同来源的爱德华氏菌中糖酵解持家酶蛋白均能分泌到胞外。【结论】爱德华氏菌中糖酵解持家酶的胞外分泌是普遍现象。     

The regulation of glycolysis in perfused locust flight muscle

Concentrations of glycolytic intermediates, amino acids and possible regulator substances were measured in extracts from locust thoracic muscles perfused under different conditions. The conversion of [(14)C]glucose into intermediates and CO(2) by muscle preparations was also followed. When muscles perfused with glucose were made anaerobic changes in metabolite concentrations occurred that could be accounted for by an activation of phosphofructokinase and pyruvate kinase. When butyrate and glucose were present in the perfusion medium the rate of glycolytic flux was lower than with glucose alone, and the aldolase reaction appeared to be inhibited. When butyrate alone was supplied to the muscle the concentrations of most glycolytic intermediates were similar to those found when glucose was supplied. Iodoacetate caused changes in concentrations of intermediates that appeared to result from inhibition of glyceraldehyde 3-phosphate dehydrogenase. Fluoroacetate-poisoned muscles showed a high citrate concentration, but no obvious site of inhibition by citrate was apparent in the glycolytic pathway. Mechanisms for control of glycolysis in locust flight muscle are discussed and related to the known properties of isolated enzymes. It is proposed that trehalase, hexokinase, phosphofructokinase, aldolase, and pyruvate kinase may be control enzymes in this tissue.     

Enzymes of glycolysis are functionally associated with the mitochondrion in Arabidopsis cells

Mitochondria fulfill a wide range of metabolic functions in addition to the synthesis of ATP and contain a diverse array of proteins to perform these functions. Here, we present the unexpected discovery of the presence of the enzymes of glycolysis in a mitochondrial fraction of Arabidopsis cells. Proteomic analyses of this mitochondrial fraction revealed the presence of 7 of the 10 enzymes that constitute the glycolytic pathway. Four of these enzymes (glyceraldehyde-3-P dehydrogenase, aldolase, phosphoglycerate mutase, and enolase) were also identified in an intermembrane space/outer mitochondrial membrane fraction. Enzyme activity assays confirmed that the entire glycolytic pathway was present in preparations of isolated Arabidopsis mitochondria, and the sensitivity of these activities to protease treatments indicated that the glycolytic enzymes are present on the outside of the mitochondrion. The association of glycolytic enzymes with mitochondria was confirmed in vivo by the expression of enolase- and aldolase-yellow fluorescent protein fusions in Arabidopsis protoplasts. The yellow fluorescent protein fluorescence signal showed that these two fusion proteins are present throughout the cytosol but are also concentrated in punctate regions that colocalized with the mitochondrion-specific probe Mitotracker Red. Furthermore, when supplied with appropriate cofactors, isolated, intact mitochondria were capable of the metabolism of (13)C-glucose to (13)C-labeled intermediates of the trichloroacetic acid cycle, suggesting that the complete glycolytic sequence is present and active in this subcellular fraction. On the basis of these data, we propose that the entire glycolytic pathway is associated with plant mitochondria by attachment to the cytosolic face of the outer mitochondrial membrane and that this microcompartmentation of glycolysis allows pyruvate to be provided directly to the mitochondrion, where it is used as a respiratory substrate.     

The association of glycolytic enzymes with cellular and model membranes

This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity     

Reconstructing the mosaic glycolytic pathway of the anaerobic eukaryote Monocercomonoides

All eukaryotes carry out glycolysis, interestingly, not all using the same enzymes. Anaerobic eukaryotes face the challenge of fewer molecules of ATP extracted per molecule of glucose due to their lack of a complete tricarboxylic acid cycle. This may have pressured anaerobic eukaryotes to acquire the more ATP-efficient alternative glycolytic enzymes, such as pyrophosphate-fructose 6-phosphate phosphotransferase and pyruvate orthophosphate dikinase, through lateral gene transfers from bacteria and other eukaryotes. Most studies of these enzymes in eukaryotes involve pathogenic anaerobes; Monocercomonoides, an oxymonad belonging to the eukaryotic supergroup Excavata, is a nonpathogenic anaerobe representing an evolutionarily and ecologically distinct sampling of an anaerobic glycolytic pathway. We sequenced cDNA encoding glycolytic enzymes from a previously established cDNA library of Monocercomonoides and analyzed the relationships of these enzymes to those from other organisms spanning the major groups of Eukaryota, Bacteria, and Archaea. We established that, firstly, Monocercomonoides possesses alternative versions of glycolytic enzymes: fructose-6-phosphate phosphotransferase, both pyruvate kinase and pyruvate orthophosphate dikinase, cofactor-independent phosphoglycerate mutase, and fructose-bisphosphate aldolase (class II, type B). Secondly, we found evidence for the monophyly of oxymonads, kinetoplastids, diplomonads, and parabasalids, the major representatives of the Excavata. We also found several prokaryote-to-eukaryote as well as eukaryote-to-eukaryote lateral gene transfers involving glycolytic enzymes from anaerobic eukaryotes, further suggesting that lateral gene transfer was an important factor in the evolution of this pathway for denizens of this environment.     

Phosphorylation of glycolytic and gluconeogenic enzymes by the insulin receptor kinase

Various glycolytic and gluconeogenic enzymes were tested as substrates for the insulin receptor kinase. Phosphofructokinase and phosphoglycerate mutase were found to be the best substrates. Phosphorylation of these enzymes was rapid, stimulated 2- to 6-fold by 10(-7) M insulin and occurred exclusively on tyrosine residues. Enolase, fructose 1,6-bisphosphatase, lactate dehydrogenases in decreasing order, were also subject to insulin-stimulated phosphorylation but to a smaller extent than that for phosphofructokinase or phosphoglycerate mutase. The phosphorylation of phosphofructokinase was studied most extensively since phosphofructokinase is known to catalyze a rate-limiting step in glycolysis. The apparent Km of the insulin receptor for phosphofructokinase was 0.1 microM, which is within the physiologic range of concentration of this enzyme in most cells. Tyrosine phosphorylation of phosphofructokinase paralleled autophosphorylation of the beta-subunit of the insulin receptor with respect to time course, insulin dose response (half maximal effect between 10(-9) and 10(-8) M insulin), and cation requirement (Mn2+ greater than Mg2+ much greater than Ca2+). Further study will be required to determine whether the tyrosine phosphorylation of phosphofructokinase plays a role in insulin-stimulated increases in glycolytic flux.     

Metabolic activity of the Sticker's lymphosarcoma

Some parameters (glycolysis, respiration, levels of glycolytic enzymes) of the lymphoid cells from the Sticker's lymphosarcoma were established in order to better define the biochemical behavior of the venereal tumor of the dog. For comparative purposes lymphocytes from peripheral blood of normal and tumor-bearing dogs were also studied. Lactic acid produced by the tumor cells during aerobic glycolysis is liberated in the reaction medium. Oxygen uptake is enhanced in the presence of succinate, but not with pyruvate, alpha-ketoglutarate, or malate as substrates. Higher levels of some of the enzymes from the glycolytic pathways as well as differences on the physicochemical and kinetic properties of the glycolytic regulatory enzymes are found in Sticker's tumor cells, when compared with the lymphocytes from peripheral blood of normal and tumor-bearing dogs. A fructose-bisphosphate positively modulated pyruvatekinase is found in the tumor cells.     

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.     

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.     

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

Summary 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.     

Clustering of sequential enzymes in the glycolytic pathway and the citric acid cycle

In recent years, evidence has been accumulating that metabolic pathways are organized in vivo as multienzyme clusters. Affinity electrophoresis proves to be an attractive in vitro method to further evidence specific associations between purified consecutive enzymes from the glycolytic pathway on the one hand, and from the citric acid cycle on the other hand. Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase. A model is presented to explain the possibility of regulation of the citric acid cycle by varying enzyme-enzyme associations between the latter three enzymes, in response to changing local intramitochondrial ATP/ADP ratios.