Showing papers on "Pyruvate kinase published in 1974"

Triglycerides and Glycerol Determination after Alkaline Hydrolysis

Abstract: Publisher Summary Triglycerides with the phosphatides, and the free and esterified cholesterol, make up the quantitatively most important lipid fractions of blood. Adipose tissue has the highest triglyceride content, between 60 and 85 %. The measurements of neutral fat by the determination of glycerol by chemical or fluorimetric methods require extraction and careful isolation of triglycerides followed by hydrolysis. In the enzymatic determination of triglycerides in blood serum or plasma, the isolation or purification of the triglycerides or the extraction of fat can be omitted. The glycerol present in serum, plasma, or Folch extracts of tissues before and after ethanolic-alkaline hydrolysis is converted to glycerophosphate and adenosine diphosphate (ADP) with ATPand glycerokinase. The ADP reacts with pyruvate kinase and added phosphoenolpyruvate to give that is reduced to lactate with NADH and lactate dehydrogenase.

Enzyme electrophoresis on cellulose acetate gel. ii. zymogram patterns in man-chinese hamster somatic cell hybrids

Abstract: Techniques are described for the electrophoretic characterization on Cellogel of adenosine deaminase, adenylate kinase, enolase, fumarase, α-galactosidase, glucosephosphate isomerase, glutathione reductase, glutamic-oxaloacetic transaminase, guanylate kinase, hexosaminidase, hypoxanthine-guanine phosphoribosyltransferase, indophenol oxidase, “malic” enzyme, mannosephosphate isomerase, nucleoside phosphorylase, peptidase-A, peptidase-B, peptidase-C, phosphoglucomutase, pyruvate kinase, triosephosphate isomerase, and UDP glucose pyrophosphorylase1 in the man-Chinese hamster somatic cell hybrid systems.

Pyruvate Kinase of Streptococcus lactis

Abstract: The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent Vmax values and decreased the Km values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The Km values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.

Mechanisms activating glycolysis in the brain in arterial hypoxia.

Abstract: — In order to study regulatory steps responsible for the activation of anaerobic glycolysis in the brain during hypoxia, cerebral concentrations of carbohydrate substrates and organic phosphates were measured in rats after reduction of the arterial PO2 to 23-25 mm Hg for 2, 5 and 15 min. The results demonstrated a progressive accumulation of lactate as well as of pyruvate and malate in the absence of changes in ATP, A DP, AMP, citrate and ammonia. The pattern of substrate changes obtained indicate that hypoxia is accompanied by activation of pyruvate kinase and of hexokinase, but not of phosphofructokinase. There was a progressive fall in intracellular pH and a moderate increase in the calculated cytoplasmic NADH/NAD+ ratio. The changes in pyruvate and in the NADH/NAD+ ratio may be responsible for the observed increase in the malate concentration.

Alterations in glucose metabolism in chick-embryo cells transformed by Rous sarcoma virus: intracellular levels of glycolytic intermediates.

Abstract: Chick-embryo cells, transformed with Rous sarcoma virus, show enhanced rates of sugar transport and glycolysis. Determination of intracellular concentrations of glycolytic intermediates suggests that the enhanced glycolytic flux is due to increased activities of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1), phosphofructokinase, (ATP:D-fructose-1-phosphate 6-phosphotransferase, EC 2.7.1.56), and pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40), and not directly to the increased glucose transport. This conclusion is supported by the finding that the intracellular concentration of free glucose is decreased, rather than increased, in the transformed cells. The present observations suggest that the increased glycolytic flux is related to an increased rate of phosphorylation of glucose, and that hexokinase in the transformed cells is at least partly released from its normal control mechanism involving feedback inhibition by glucose-6-P.

Influence of insulin on growth and metabolism of 7,12-dimethylbenz(alpha)anthracene-induced mammary tumors.

Abstract: Summary The influence of insulin on growth and carbohydrate metabolism of 7,12-dimethylbenz(a)anthracene- (DMBA) induced mammary tumors was examined. Approximately 60% of the DMBA-induced tumors regressed after the animals were made diabetic by streptozotocin treatment, a pattern of tumor growth that was reversed by administration of 2 IU of insulin per day to diabetic rats. DMBA-induced tumors regressing in diabetic animals, termed insulin dependent, demonstrated significant decreases in the activities of pyruvate kinase, phosphofructokinase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. Tumors that continued to grow in diabetic rats, termed insulin independent, showed enzyme activities similar to those of growing tumors in intact animals. Examination of utilization of labeled glucose in vitro by slices of tumors regressing in diabetic rats indicated that there was a decrease in both the pentose phosphate pathway and the glycolytic pathway, on the basis of 14CO2 production and incorporation into fatty acids of labeled glucose substrate. Thus, tumor regression and reduced carbohydrate metabolism appear to be correlated among those carcinomas that have been termed insulin dependent, suggesting that insulin should be considered as another hormonal factor in DMBA-induced breast cancers.

Pyruvate Kinase Assay in Serum and Erythrocytes

Abstract: Publisher Summary This chapter elaborates on pyruvate kinase (PK), which was discovered by Lohmann and Meyerhof and in 1942 first crystallized from rat muscle by Negelein. In the rat, the PKs from skeletal muscle, heart muscle, and brain differ from those from liver and kidney. In nonsphereocytic hemolytic anemia, a deficiency of PK in the erythrocytes was established. The disease is genetically determined and appears to be autosomal recessive. PK is applied in biochemistry and clinical chemistry. Lactate dehydrogenase (LDH) must not contain more than 0.01% PK; the Boehringer preparation conforms to this requirement. As the reaction is started with adenosine-5′-diphosphate (ADP) solution, all interfering compounds can react before the PK reaction and therefore, the PK assay is specific.

Delayed in vitro utilization of glucose by Leishmania tropica promastigotes.

Abstract: SYNOPSIS Leishmania tropica promastigotes do not utilize glucose provided in the medium until late log phase. Rapid depletion of glucose from the medium, however, occurs during late log and stationary phases. At about the same time, the cells show maximal rates of glucose uptake as well as peak levels of phosphofructokinase and pyruvate kinase activities. The glucose analog, 2-deoxy-D-glucose inhibits glucose transport. Incorporation of this analog in the growth medium results in inhibition of growth. The hexokinase of L. tropica phosphorylates 2-deoxy-D-glucose. Pyruvate kinase is activated by fructose-1, 6-diphosphate and adenosine monophosphate.

Serum pyruvate kinase in muscle disease and carrier states

Abstract: The incidence of elevated serum pyruvate kinase levels is compared with that of creatine phosphokinase levels in 29 patients with muscular dystrophy and in 26 carriers of Duchenne muscular dystrophy. The incidence of elevated pyruvate kinase values in the dystrophic cases is 50 percent higher than the creatine phosphokinase values, Comparison of these serum enzyme levels in the carrier state show the pyruvate kinase test to be abnormally elevated in 15 cases and the creatine phosphokinase in five cases. A broad screening study is required to support and expand these provocative results.

Competition for ADP between Pyruvate Kinase and Mitochondrial Oxidative Phosphorylation as a Control Mechanism in Glycolysis

Abstract: To assess a possible role of pyruvate kinase as a site for respiratory and glycolytic interaction, competition for ADP between pyruvate kinase and respiratory phosphorylation was measured in a model system consisting of rat liver mitochondria respiring in the presence of pyruvate kinase, phosphoenolpyruvate, ATP, and an ADP-regenerating system consisting of glucose and purified yeast hexokinase. This system allowed determination of total ATP production, equivalent to ADP utilization, by measuring glucose 6-phosphate formation; ADP utilization by pyruvate kinase by pyruvate formation; and respiratory ADP utilization by Pi uptake. O2 uptake was measured by means of an oxygen electrode. In the presence of a respiratory substrate such as succinate or glutamate-malate, the addition of pyruvate kinase and phosphoenolpyruvate reduced O2 uptake as well as oxidative phosphorylation about 80%. Respiration was increasingly inhibited with increasing pyruvate kinase, and this inhibition was decreased with increased hexokinase or ATP. Mitochondrial respiratory inhibition by pyruvate kinase and phosphoenolpyruvate was accompanied by an increase in the ATP/ADP ratio from 0.3 to 32. These inhibitory effects of pyruvate kinase on respiration were abolished by addition of 2,4-dinitrophenol. These results are in acccord with a role of pyruvate kinase as a determinant of glycolytic activity by competing with oxidative phosphorylation for the available ADP; and provide additional support for our previous suggestion that high glycolysis of certain tumors may be attributable to their extraordinarily high pyruvate kinase activity.

Pyruvate kinase from the mantle tissue of (itMytilus Edulis) L.

Abstract: 1. 1. Mantle pyruvate kinase (E.C.2.7.1.40) is subject toa allosteric regulation, being inhibited by l -alanine and ATP and activated by fructose- 1,6-diphosphate (FDP). 2. 2. Substrate dependence of the enzyme is sigmoidal, and the sigmoidicity is increased at low pH. 3. 3. FDP at physiological concentrations overrides inhibition of the enzyme by l -alanine. 4. 4. The result of these modulators acting together suggests that both the reaction catalysed by pyruvate kinase (PEP → pyruvate) and the reaction catalysed by phosphoenolpyruvate carboxykinase (PEP → oxoloacetate) could proceed together, with the predominancer of one reaction over the other being determined by the degree of tissue hypoxia. 5. 5. The seasonal changes in the enzyme-substrate affinity are consistent with the known seasonal glycolytic and gluconeogenic sequence in the mantle.

Anaerobic excretion of fermentation acids by Hymenolepis diminuta during development in the definitive host.

Abstract: Succinic, acetic, and lactic acids are the major anaerobic fermentation acids excreted in vitro by Hymenolepis diminuta. In parasites recovered from immature (6-day) infections in white rats that were fed ad lib. in the dark for 12 hr, approximately equal amounts of succinic and lactic acids, and less acetic acid, were excreted. In the succeeding 12-hr period of fasting in the light, production of succinic and acetic acids from endogenous glycogen reserves was unaffected, whereas that of lactic acid decreased markedly. The pattern was different in parasites recovered from 10and 14-day infections, in that quantitative differences in acid excretion during feeding and fasting periods were not observed, lactic acid production was relatively small, and succinic acid constituted about half of the total excreted acids. An active pyruvate decarboxylase enzyme complex presumably accounted for the formation of acetate from pyruvate. The existence of lactate dehydrogenase, and a phosphoenolpyruvate carboxykinase leading to succinate production were already known. A calculation of the carbon content of excreted acids in terms of ,moles phosphoenolpyruvate carbon/mg protein showed that the rates of excretion of fermentation acids in 6and 14-day infections did not differ. Succinic, acetic, and lactic acids comprise most of the total fermentation acids excreted in vitro by Hymenolepis diminuta recovered from patent (15 day) infections in rats (Fairbairn et al., 1961). It is known that phosphoenolpyruvate is a key intermediate in the formation of these acids since it reacts with carbon dioxide in the presence of phosphoenolpyruvate carboxykinase and GDP or IDP to form oxaloacetate and thence succinic acid, and is also converted to pyruvate by pyruvate kinase (Bueding and Saz, 1968) and hence to L(+) lactate by lactate dehydrogenase (Walkey and Fairbairn, 1973). Acetic acid is presumably formed by oxidative decarboxylation of pyruvate, although the existence of this enzyme complex has not been studied. Different isoenzymes of L(+) -lactate dehydrogenase occur in the anterior regions of the mature tapeworm strobila or in immature (6 day) parasites, and in infective eggs obtained from mature proglottids (Walkey and Fairbairn, 1973). Similar observations have been made on pyruvate kinase (Carter and Fairbairn, unpublished). There is reason to believe, therefore, that quantitative differences in the forReceived for publication 11 February 1974. * This work was supported by Grants AI-08491 and 5 TOI AI-226 from the NIH, Bethesda, Maryland 20014. t Present address: The Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, England. mation and excretion of succinic, acetic, and lactic acids may exist in different regions of the strobila, or in parasites at different stages of development in the definitve host. This is not surprising, as the strobila of H. diminuta contains hundreds of proglottids representing all stages of continuous growth and differentiation. In the present investigation we have compared the in vitro anaerobic excretion of succinic, acetic, and lactic acids by H. diminuta recovered from infected rats after 6 and 14 days, respectively, and have demonstrated the decarboxylation of pyruvate by a pyruvate decarboxylase enzyme complex. MATERIALS AND METHODS In order to make possible a comparison of the present experiments with other recently completed experiments (Carter and Fairbairn, unpublished), white, male Sprague-Dawley rats (Holtzman strain) weighing about 150 g each were infected with 30 cysticercoids of H. diminuta injected into the stomach through a catheter. After ensuring that all cysticercoids had been delivered from the catheter, the rats were caged in pairs and maintained on a 12 hr light-12 hr dark daily photoperiod. Food (commercial pellets) was provided only during the dark half of the cycle, from 8:00 PM to 8:00 AM. Rats harboring parasites to be recovered on the 10th or 14th day after infection were maintained in this manner. When parasites were to be recovered after infection for 6 days the procedure was the same except that greater numbers of rats were infected, each with 300 to 500 cysticercoids, in order that sufficient weight of tissue would be available for

Interrelationship between nitrate assimilation and carbohydrate metabolism in plant roots.

Abstract: The effect of nitrate incubation on the pattern of carbohydrate metabolism in different regions of the pea (Pisum sativum L. var. Kelvedon Wonder) root has been studied. Roots were incubated in a 10 mM potassium nitrate solution for 4, 8 and 12 h. Marked increases were noted in the activities of nitrate assimilation enzymes after 4 h. Increased activities were also recorded for hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and transketolase. No consistent changes were observed in the activities of phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. Experiments with [1-(14)C] and [6-(14)C]glucose indicated a relative shift in the pattern of carbohydrate oxidation from glycolysis to the pentose phosphate pathway. The data are interpreted as indicating a close interrelationship between nitrate assimilation and carbohydrate metabolism, particularly in relation to the supply of NADPH by the pentose phosphate pathway for nitrite reductase.

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers.

Abstract: Kinetic properties of rat liver pyruvate kinase type I at pH75 and 65 were studied with physiological ranges of substrates, modifiers and Mg2+ concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx 01mg/ml) Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH75 The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate Decreasing the pH to 65 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly