Showing papers on "Glycolysis published in 1969"

The kinetic properties of citrate synthase from rat liver mitochondria.

Abstract: 1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.

Hypoglycin and hypoglycin-like compounds

Abstract: Hypoglycin and hypoglycin-like compounds cause profound hypoglycemia which may be largely attributed to their effects on gluconeogenesis. The toxicity of these agents is due to their capacity to become activated to acyl CoA derivatives whose further oxidation is impaired. Because the activated compounds serve as substrates for the carnitine acetyltransferase, tissue levels of both free carnitine and coenzyme A are depressed. The depression of these cofactors results in a decrease in long-chain fatty acid oxidation, whose products (acetyl CoA, NADH, ATP) are necessary for gluconeogenesis. Moreover, the decreased oxidation of long-chain fatty acids may also result in augmented glycolysis due to relief of the inhibitory effects of long-chain fatty acid oxidation on glycolysis. Replacement of coenzyme A and carnitine prevent inhibition of long-chain fatty acid oxidation and glyconeogenesis by 4-pentenoate. Addition of noncarnitinedependentfatty acids or palmitylcarnitine as substrates also partially prevent the 4-pentenoate depression of gluconeogenesis. The inability to completely reverse the toxic effects of these compounds in vitro and in vivo suggests that they may be exerting as yet poorly established effects on oxidative phosphorylation and the electron transport system.

Glycerol kinase activities in muscles from vertebrates and invertebrates

Abstract: 1. Glycerol kinase (EC 2.7.1.30) activity was measured in crude extracts of skeletal muscles by a radiochemical method. The properties of the enzyme from a number of different muscles are very similar to those of the enzyme from rat liver. Glycerol kinase from locust flight muscle was inhibited competitively by l-3-glycerophosphate with a Ki of 4·0×10−4m. 2. The activity of glycerol kinase was measured in a variety of muscles from vertebrates and invertebrates in an attempt to explain the large variation in the activity of this enzyme in different muscles. 3. In vertebrates glycerol kinase activities were generally higher in red muscle than in white muscle; the highest activities (approx. 0·2μmole/min./g. fresh wt.) were found in the red breast muscle of some birds (e.g. pigeon, duck, blue tit) whereas the activities in the white breast muscle of the pheasant and domestic fowl were very low (approx. 0·02μmole/min./g.). 4. On the basis of glycerol kinase activities, muscles from insects can be classified into three groups: muscles that have a low enzyme activity, i.e. 1·5μmoles/min./g. (e.g. bees, wasps, some blowflies). 5. The function of glycerol kinase in vertebrate and insect muscles that possess a low or intermediate activity is considered to be the removal of glycerol that is produced from lipolysis of triglyceride or diglyceride by the muscle. Therefore in these muscles the activity of glycerol kinase is related to the metabolism of fat, which is used to support sustained muscular activity. A possible regulatory role of glycerol kinase in the initiation of triglyceride or diglyceride lipolysis is discussed. 6. The function of glycerol kinase in the insect muscles that possess a high activity of the enzyme is considered to be related to the high rates of glycolysis that these muscles can perform. The oxidation of extramitochondrial NADH, and therefore the maintenance of glycolysis, is dependent on the functioning of the glycerophosphate cycle; if at any stage of flight (e.g. at the start) the rate of mitochondrial oxidation of l-3-glycerophosphate was less than the activity of the extramitochondrial glycerophosphate dehydrogenase, this compound would accumulate, inhibit the latter enzyme and inhibit glycolysis. It is suggested that such excessive accumulation of l-3-glycerophosphate is prevented by hydrolysis of this compound to glycerol; the latter would have to be removed from the muscle when the accumulation of l-3-glycerophosphate had stopped, and this would explain the presence of glycerol kinase in these muscles and its inhibition by l-3-glycerophosphate.

Enzymes Involved in Fructose Metabolism in Liver and the Glyceraldehyde Metabolic Crossroads

Abstract: quantitative model of the metabolic crossroads of D-glyceraldehyde has been developed. This model permits a prediction of the relative contributions of the phosphorylative, oxidative and reductive pathways in different conditions. The main pathway for D-glyceraldehyde metab- olism in liver is phosphorylation by triokinase. Many investigations have been directed to look for the pathway(s) of incorporation of fructose into the common glycolytic pathway.

The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells.

Abstract: 1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The Km values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and Pi found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [14C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the 14CO2 yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.

Oxygen-hemoglobulin dissociation curves: effect of inherited enzyme defects of the red cell.

Abstract: The blood of a patient with a deficiency of hexokinase in the red cells and a decreased concentration of 2, 3-diphosphoglycerate in the red cells showed an increased affinity for oxygen, whereas a patient with a deficiency of pyruvate kinase and an elevated concentration of 2, 3-diphosphoglycerate in the red cells had blood with a decreased affinity for oxygen. Defects in red cell glycolysis may alter the oxygen affinity of blood by virtue of their effect on 2, 3-diphosphoglycerate concentrations in red cells.

Effects of Colicins E1 and K on Cellular Metabolism

Abstract: Colicins E1 and K inhibited a whole series of energy-dependent reactions in Escherichia coli cells, including motility, biosynthesis of nucleic acids, proteins and polysaccharides, and the conversion of ornithine to citrulline. Respiration was only partially affected, and substrates such as glucose continued to be catabolized through the normal pathways, albeit with reduced CO2 production. The soluble products of aerobic glucose catabolism by colicin-treated cells were analyzed. Pyruvate replaced acetate as the major excreted product, and the following intermediates of glycolysis were excreted in significant amounts: glucose-6-phosphate, fructose-1,6-diphosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate. Anaerobically growing cells manifested a somewhat enhanced tolerance to the colicins. This protection by anaerobiosis appeared to depend on the exclusion of oxygen more than on the extent of fermentative catabolism versus catabolism of the respiratory type. These results are interpreted in terms of possible functions of colicin in lowering the adenosine triphosphate (ATP) content of the cells and in terms of the role of lowered ATP levels in inhibiting many of the energy-requiring reactions.

Adenosine Triphosphate Levels of Mammalian Pancreatic B Cells after Stimulation with Glucose and Hypoglycemic Sulfonylureas

Abstract: A microchemical technic was applied to elucidate the possible role of ATP in insulin secretion by measuring the levels of this metabolite in pancreatic islets from obese hyperglycemic mice. The β cell content of ATP was markedly reduced within the first minute after interruption of the blood supply. A steady-state level of about 5 mmoles of ATP was noted in islets incubated in the absence of glucose. The corresponding ATP level was twice as high when at least 1 mg./ml. of glucose was present in the incubation medium. While the contents of ATP and glycogen remained constant when the pancreatic islets were incubated with diazoxide, the amounts of both these metabolites were significantly reduced by concentrations of sulfonylurea compounds known to stimulate the insulin release. The sulfonylurea effect on the islet ATP content implies a change in the “phosphate potential” of the β cells, which might stimulate glycogenolysis and increase the glycolytic flux. The sulfonylurea stimulation of insulin release might thus be related to a product of glucose degradation beyond the level of glucose-6-phosphate.

Properties and mechanism of action of pyruvate, phosphate dikinase from leaves.

Abstract: 1. Sugar-cane leaf pyruvate,P(i) dikinase was prepared free of enzymes that would interfere with studies on the stoicheiometry and mechanism of the reaction it catalyses. The reaction was unequivocally shown to involve the conversion of equimolar amounts of pyruvate, ATP and P(i) into phosphoenolpyruvate, AMP and PP(i). 2. The purified enzyme was stable at pH8.3 only if stored at about 20 degrees in the presence of Mg(2+) and a thiol-reducing reagent, care being taken to prevent the oxidation of the thiol. 3. The apparent Michaelis constants for phosphoenolpyruvate and PP(i) were 0.11mm and 0.04mm respectively and that for AMP was less than 4mum. 4. At pH8.3 the initial velocity of the reaction was about 6 times as fast in the direction towards phosphoenolpyruvate synthesis as in the reverse direction. 5. With the exception of ATP, all the products of the reaction in both directions were inhibitory. 6. The phosphate groups of PP(i) were derived from P(i) and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps:Enzyme+ATP+P(i) right harpoon over left harpoon Enzyme-P+AMP+PP(i)Enzyme-P+pyruvate right harpoon over left harpoon Enzyme+phosphoenolpyruvate

Regulation of the Level of Key Enzymes of Glycolysis and Gluconeogenesis in Liver

Abstract: pyruvate carboxykinase were low. A metabolic bifurcation to glycolysis and gluconeogenesis, below and above the trioscphosphates level respectively, has then been shown to be easily maintained in liver. It is suggested that the induction of glucokinase and pyruvate kinase in diabetic animals after insulin administration is a sequential process that involves hormonal induction of glucokinase by insulin and secondary metabolite induction of the L isoenzyme of pyruvate kinase by some glycolytic intermediate. The occurrence of independent mechanisms for the regulation in liver of the activity of enzymes which catalyze irreversible steps of glycolysis and gluconeogenesis affords a valuable metabolic plasticity. Changes in the apparent concentration of a number of enzymes in liver have been observed in relation to the nutritional and hormonal conditions of the animal. Some of these changes could be of considerable significance in the regulation of major metabolic pathways. Glucose-6-phosphatase [l], fructosediphosphatase [a], phosphoenolpyruvate carboxykinase [3] and pyruvate carboxylase [4] had been reported to be increased in gluconeogenic conditions, like fasting, diabetes, or corticosteroids administration. In 1963, an insulin-dependent glucokinase was identified [5-71. After the finding of the induction of glucokinase in rat liver by insulin, Weber et al. [S] postulated the hypothesis that the key steps of glycolysis and gluconeogenesis could depend on two functional genic units, with insulin as inducer of the glycolytic unit and repressor of the gluconeogenic one. Shortly afterwards a number of observations were reported on the behaviour of pyruvate kinase [9- 111 and phosphofructokinase [12], consistent with the hypothesis of induction by insulin of the enzymes that catalyze these two irreversible steps of glycolysis in liver. This paper presents the results of a nutritional approach involving diets rich in fructose and/or

The effect of dietary and hormonal conditions on the activities of glycolytic enzymes in rat epididymal adipose tissue.

Abstract: 1. Measurements were made of the activities of nine glycolytic enzymes in epididymal adipose tissues obtained from rats that had undergone one of the following treatments: starvation; starvation followed by re-feeding with bread or high-fat diet; feeding with fat without preliminary starvation; alloxan-diabetes; alloxan-diabetes followed by insulin therapy. 2. In general, the activities of the glycolytic enzymes of adipose tissue, unlike those of liver, were not greatly affected by the above treatments. 3. The ;key' glycolytic enzymes, phosphofructokinase and pyruvate kinase, were generally no more adaptive in response to physiological factors than other glycolytic enzymes such as glucose phosphate isomerase, fructose diphosphate aldolase, triose phosphate isomerase, glycerol 3-phosphate dehydrogenase, phosphoglycerate kinase and lactate dehydrogenase. 4. Adiposetissue pyruvate kinase did not respond to feeding with fat in a manner similar to the liver enzyme. 5. Glyceraldehyde phosphate dehydrogenase had a behaviour pattern unlike the other eight glycolytic enzymes studied in that its activity was depressed by feeding with fat and was not restored to normal by re-feeding with a high-fat diet after starvation. These results are discussed in relation to the requirements of adipose tissue for glycerol phosphate in the esterification of fatty acids. 6. A statistical analysis of the results permitted the writing of linear equations describing the relationships between the activities of eight of the enzymes studied. 7. Evidence is presented for the existence of two constant-proportion groups amongst the enzymes studied, namely (i) glucose phosphate isomerase, phosphoglycerate kinase and lactate dehydrogenase, and (ii) triose phosphate isomerase, fructose diphosphate aldolase and pyruvate kinase. 8. Mechanisms for maintaining the observed relationships between the activities of the enzymes in the tissue are discussed.

Hemolytic anemia with impaired hexokinase activity

Abstract: Analyses of key glycolytic intermediates in freshly drawn red cells from six related individuals suggest that decreased hexokinase activity underlies the hemolytic process in the two members with overt hemolysis. Low red cell glucose 6-phosphate (G6P) was observed not only in the anemic patients but in the presumptive heterozygotes as well and served as a useful marker for the presence of the trait. Hexokinase activity was labile in distilled water hemolysates but was only slightly low when protected by glucose, mercaptoethanol, and ethylenediaminetetraacetate (EDTA). Normal red cell hexokinase was demonstrated to be dependent on glucose for maintenance of activity after heating to 45 degrees C. The cells of the proposita are unable to utilize glucose efficiently at glucose concentrations lower than 0.2 mmole/liter whereas normal cells maintain linear glucose consumption to at least 0.05 mM glucose. These qualitative abnormalities could result from the presence of a mutant hexokinase with an abnormally reactive sulfhydryl group and altered substrate affinity in the red cells of this kindred.

Islet-cell metabolism during insulin release. Effects of glucose, citrate, octanoate, tolbutamide, glucagon and theophylline

Abstract: 1. Concentrations of glucose 6-phosphate and 6-phosphogluconate were studied in islets of Langerhans isolated from rat pancreas and incubated in the presence of various agents that induce insulin release. 2. In response to rising concentrations of extracellular glucose (2–10mm) there is a linear increase in the intracellular concentration of glucose 6-phosphate, though this is not the case for 6-phosphogluconate, the intracellular concentration of which only increases when the external glucose concentration exceeds 5mm. 3. Tolbutamide, octanoate and citrate, all of which promote insulin secretion from isolated islets, increase the intracellular concentrations of glucose 6-phosphate and 6-phosphogluconate. The results obtained in the presence of octanoate and citrate are compatible with an inhibitory effect of citrate on islet-cell phosphofructokinase. 4. Theophylline and glucagon when incubated with islets in vitro promote insulin release and cause a rise in 6-phosphogluconate concentration and not in that of glucose 6-phosphate. 5. It is suggested that the further metabolism of glucose 6-phosphate through a pathway other than glycolysis is essential for insulin release. One such pathway involves its oxidation to 6-phosphogluconate, which seems to be a necessary accompaniment of insulin secretion due to glucose. The possibility that agents other than glucose promote insulin release by enhancing the oxidation of glucose 6-phosphate through this pathway is discussed.

Red cell metabolism in the newborn infant. v. glycolytic intermediates and glycolytic enzymes

Abstract: Previous studies demonstrated that the erythrocytes of the newborn infant have a relative phosphofructokinase deficiency and inappropriate glucose consumption for cell age. In view of these findings, an analysis of red cell glycolytic intermediates and all Embden-Meyerhof pathway enzyme activities was made in the red cells of infants, normal adults, and subjects with reticulocytosis to define further differences in metabolism and search for evidence of a metabolic blockade. The erythrocytes of subjects with reticulocytosis were found to possess increased quantities of glucose-6-phosphate, fructose-6-phosphate and 2,3-diphosphoglycerate. The elevation of the hexose phosphates was even greater in the red cells of the premature infants and was associated with a decrease in levels of 2,3-diphosphoglycerate and phosphoenolpyruvate. Measurement of enzyme activities indicated that, in addition to the phosphofructokinase deficiency, the cells of the newborn infant have markedly increased activities of enolase and phosphoglycerate kinase. It is suggested that the increase in the level of the hexose phosphates in the erythrocytes of the infants and subjects with reticulocytosis is a reflection of this relative phosphofructokinase deficiency, with this deficiency being greater in newborn infants. The differences in the enzymatic profile between the erythrocytes of the newborn and those of the normal adult is not solely a function of their younger age. Some of the differences are a reflection of the presence of a cell unique to this period of life.

Adenine nucleotides and magnesium ions in relation to control of mammalian cerebral-cortex hexokinase

Abstract: 1. The kinetics of inhibition of brain soluble cytoplasmic hexokinase by ADP were examined in relation to variations in the concentrations of Mg(2+) and ATP. The type of inhibition observed was dependent on the Mg(2+)/ATP ratio. 2. ADP at Mg(2+)/ATP ratios 2:1 exhibited inhibition of the ;mixed' type; at Mg(2+)/ATP ratios 1:1 the inhibition appeared to be competitive with regard to ATP. 3. Inhibition by free ATP was observed when the Mg(2+)/ATP ratio was less than 1:1. The inhibition was also of the ;mixed' type with respect to MgATP(2-). 4. The inhibitions due to ADP and to free ATP were not additive. The results suggested that there may be up to four sites in the soluble enzyme: for glucose, glucose 6-phosphate, ADP and MgATP(2-). 5. The ;free' non-particulate intracellular Mg(2+) concentration was measured and concluded to be about 1.5mm. 6. The concentrations in vivo of Mg(2+) and ATP likely to be accessible to a cytoplasmic enzyme are suggested to be below those that yield maximum hexokinase rates in vitro. The enzymic rates were measured at relevant suboptimum concentrations of Mg(2+) and ATP in the presence of ADP. Calculations that included non-competitive inhibition due to glucose 6-phosphate (56-65% at 0.25mm) resulted in net rates very similar to the measured rates for overall glycolysis. This system may therefore provide a basis for effective control of cerebral hexokinase.

Purification and properties of sheep liver phosphofructokinase.

Abstract: 1. The activity of phosphofructokinase in sheep liver was found to be dependent on the composition and molarity of the buffer used in extraction. Under optimum conditions a value of 4–7μmoles/min./g. wet wt. of tissue was obtained. 2. The enzyme was purified 480-fold by a combination of ammonium sulphate fractionation, heat treatment in the presence of ethanol, DEAE-cellulose chromatography and Sephadex G-200 gel filtration. The final specific activity was 18·5μmoles/min./mg. of protein. 3. The purified enzyme was inhibited by ATP and citrate, the degree of inhibition depending on the concentration of fructose 6-phosphate, magnesium chloride and ammonium sulphate, as well as on the pH. ATP and citrate inhibition was overcome by AMP and fructose 1,6-diphosphate. 4. The enzyme was also inhibited by NADH and NADPH in a manner largely independent of other components of the assay medium. AMP and fructose 1,6-diphosphate were not able to overcome this type of inhibition. 5. Octanoate was not an inhibitor of phosphofructokinase. 6. Differences between these results and those of other workers are discussed.

Influence of Glucose on the Transmembrane Action Potential of Guinea-Pig Papillary Muscle: Metabolic Inhibitors, Ouabain, and Calcium Chloride, and their Interaction with Glucose, Sympathomimetic Amines, and Aminophylline

Abstract: In a previous report, we have proposed that anaerobic glycolysis alone can maintain an action potential of normal duration in guinea-pig papillary muscle. This proposal followed from the observation that 50 mM glucose, several sympathomimetic amines, and aminophylline all caused the duration of the action potential of muscles, which had been reduced by incubation in a medium containing 5 mM glucose, to return toward control values. This occurred in the presence or absence of oxygen. It was further proposed that the ATP resulting from anaerobic glycolysis was in some way preferentially utilized in the membrane by a process controlling repolarization and therefore the duration of the action potential. The present experiments were undertaken to determine what metabolic pathways were necessary for the glucose effect mentioned above, by the use of the metabolic inhibitors sodium cyanide, iodoacetic acid, and 2,4-dinitrophenol. Secondly, the effects of ouabain and calcium, which are known to modulate the utilization of ATP, on the glucose effect were studied. It was concluded that an intact glycolytic pathway is necessary for the effect of glucose on the electrical activity of papillary muscle and that the effect can be increased or decreased by low or high doses of ouabain, respectively. The actions of ouabain are thought to be due to a stimulation and depression of a membrane ATP-ase.