Showing papers on "Pyruvate kinase published in 1968"
197 citations
TL;DR: It is concluded that enzyme-substrate and enzyme-modulator interactions are important factors in short-term and in evolutionary adaptations by poikilotherms to changes in temperature.
Abstract: 1. The effects of temperature on the catalytic and regulatory properties of pyruvate kinases from the temperate-zone rainbow trout and the Antarctic fish Trematomus bernacchii were examined. 2. The K(m) value of pyruvate kinase for one of its two substrates, phosphoenolpyruvate, is temperature-dependent, and is lowest at temperatures that closely coincide with the habitat temperatures of the two fishes. 3. Two regulatory functions of pyruvate kinase, feedforward activation by fructose diphosphate and feedback inhibition by ATP, are temperature-independent. Enzyme-ADP interaction is also temperature-independent. 4. It is concluded that enzyme-substrate and enzyme-modulator interactions are important factors in short-term and in evolutionary adaptations by poikilotherms to changes in temperature. Though the K(m) for substrate may vary in apparently adaptive manners, the regulatory functions of an enzyme appear to be unchanged over the range of temperatures experienced by the organism in Nature.
164 citations
TL;DR: Marked differences in pyruvate kinase activities were found to occur in three parasitic helminths; in Ascaris muscle the avtivity of this enzyme was 100–200 times lower than in Schistosoma mansoni and 10 times higher than in the tapeworm Hymenolepis diminuta.
158 citations
145 citations
TL;DR: The pyruvate-phosphate ligase (AMP) as discussed by the authors is a pyrolytica trophozoite enzyme that catalyzes the reversible transformation of phosphoenolpyruvates to pyruVate according to the following equation: P-enolpyrevate + AMP + PPi ⇄ pyruveate + ATP + Pi
111 citations
TL;DR: It is found that 13-glucosides are accumulated solely as phosphorylated derivatives and present evidence that the sequence of reactions leading to 83- glucoside catabolism is as follows.
Abstract: Two distinct mechanisms catalyzing glucoside uptake have been detected in E. coli K12. One of these has high stereospecificity for glucose and alkyl-aor alkyl-,3-glucosides,l and the second, for alkyl-jBand aryl-,3-glucosides.2' 3 Lin and his collaborators4 5 have concluded that aMG is not actively transported, but is trapped intracellularly as the result of a phosphorylation reaction catalyzed by enzymes of the kinase system of Kundig, Ghosh, and Roseman.' Schaefler reported that ethyl-1-thio-f3-glucoside (TEG) was present intracellularly as a phosphorylated derivative when uptake was mediated by the mechanism responsible for aMG accumulation, but as the free glycoside when accumulated by the mechanism eliciting high specificity for aryl-f3-glucosides in a mutant which did not accumulate aMG.2 He concluded that the accumulation of TEG by cells in which both mechanisms for glucoside uptake were operative was mediated primarily by the one which catalyzed uptake of TEG in nonphosphorylated form. This conclusion implies that uptake by the phosphorylating mechanism does not involve equilibration of unphosphorylated substrates across the cell membrane, for such equilibration would allow the actively transported glucoside to exit. In contrast to Schaefler's observations, we find that 13-glucosides are accumulated solely as phosphorylated derivatives and present evidence that the sequence of reactions leading to 83-glucoside catabolism is as
108 citations
TL;DR: The activities of those enzymes involved in the conversion of glucose into lipid are low during the neonatal period and increase with weaning, and NADP-specific malate dehydrogenase first appears and develops at the beginning of the weaning period.
Abstract: 1. The activities of some enzymes involved in both the utilization of glucose (pyruvate kinase, ATP citrate lyase, NADP-specific malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP-specific isocitrate dehydrogenase, all present in the supernatant fraction of liver homogenates) and the formation of glucose by gluconeogenesis (glucose 6-phosphatase in the whole homogenate and fructose 1,6-diphosphatase, phosphopyruvate carboxylase, NAD-specific malate dehydrogenase and fumarase in the supernatant fraction) have been determined in rat liver around birth and in the postnatal period until the end of weaning. 2. The activities of those enzymes involved in the conversion of glucose into lipid are low during the neonatal period and increase with weaning. NADP-specific malate dehydrogenase first appears and develops at the beginning of the weaning period. 3. The marked increase in cytoplasmic phosphopyruvate carboxylase activity at birth is probably the major factor initiating gluconeogenesis at that time. 4. The results are discussed against the known changes in dietary supplies and the known metabolic patterns during the period of development.
100 citations
TL;DR: Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu(2+) inhibition, little evidence of significant reversal of the inhibition by these compounds could be found.
Abstract: 1. Preincubation of partially purified rat liver L-type pyruvate kinase at 25° for 10min. causes a marked increase in co-operativity with respect to both the substrate, phosphoenolpyruvate, and the allosteric activator, fructose 1,6-diphosphate. 2. The results are consistent with the existence of two forms of liver L-type pyruvate kinase, designated forms LA and LB. It is postulated that form LA has a low Km for phosphoenolpyruvate (about 0·1mm) and is not allosterically activated, whereas form LB is allosterically activated by fructose 1,6-diphosphate, exhibiting in the absence of the activator sigmoidal kinetics with half-maximal activity at about 1mm-phosphoenolpyruvate. In the presence of fructose 1,6-diphosphate, form LB gives Michaelis–Menten kinetics with Km less than 0·1mm. It is further postulated that preincubation converts form LA into form LB. 3. The influence of pH on the preincubation effect was studied. 4. The inhibition of pyruvate kinase by Cu2+ was studied in detail. Though phosphoenolpyruvate and fructose 1,6-diphosphate readily protect the enzyme against Cu2+ inhibition, little evidence of significant reversal of the inhibition by these compounds could be found. 5. The effects of starvation, fructose feeding and preincubation on the pyruvate kinase activity of crude homogenates of various tissues of the rat were also studied.
94 citations
TL;DR: The regulatory properties of pyruvate kinase from Escherichia coli were investigated and fructose 1,6-diphosphate and AMP were found to activate the enzyme, but these two positive effectors had fundamentally different effects on the sigmoidal plots of rate-concentration data.
91 citations
TL;DR: Attempts at partial purification have revealed that the enzyme resembles the pyruvate kinase of skeletal muscle, rather than that of liver, in its solubility in ammonium sulphate and elution from DEAE-cellulose.
Abstract: 1. Extraction of rat epididymal adipose tissue with buffer containing EDTA yields a pyruvate kinase, provisionally called PyK-A, the properties of which resemble in several respects those of the allosteric pyruvate kinase of liver. These properties include co-operative interactions with phosphoenolpyruvate, Mg(2+), K(+), NH(4) (+) and ATP, and sensitivity to activation by fructose 1,6-diphosphate. 2. Extraction in the absence of EDTA yields predominantly a form, PyK-B, that shows both normal Michaelis-Menten kinetics with phosphoenolpyruvate, Mg(2+) and ATP, and co-operative interactions with K(+) and NH(4) (+); this form is insensitive towards fructose 1,6-diphosphate. 3. Both forms yield simple kinetics with ADP, though K(m) values differ in the two systems. In all cases where co-operativity has been demonstrated, Hill-plot n values are between 1.4 and 2.0. 4. The conversion of PyK-A into PyK-B is mediated specifically by fructose 1,6-diphosphate; the reverse reaction is occasioned by EDTA, ATP or citrate. It is thought that a bivalent cation may be involved in this interconversion. 5. Attempts at partial purification have revealed that the enzyme resembles the pyruvate kinase of skeletal muscle, rather than that of liver, in its solubility in ammonium sulphate and elution from DEAE-cellulose. 6. The relevance of these properties in the regulation of pyruvate kinase activity in vivo in adipose tissue is discussed.
85 citations
TL;DR: The Longissimus dorsi muscles from Chester White, Hampshire and Poland China animals were used to establish certain differences in metabolic intermediate patterns between muscles with “fast” and “slow” rates of post-mortem glycolysis, and Adenine nucleotide levels appeared to be the primary regulatory factors for phosphorylase.
Abstract: SUMMARY— The Longissimus dorsi muscles from Chester White, Hampshire and Poland China animals were used to establish certain differences in metabolic intermediate patterns between muscles with “fast” and “slow” rates of post-mortem glycolysis. Metabolic intermediate patterns were consistent with the concept that phosphorylase is the primary control site of postmortem glycolysis. Adenine nucleotide levels appeared to be the primary regulatory factors for phosphorylase. The phosphofructokinase and pyruvate kinase enzymes were also involved in post-mortem glycolytic control. Levels of high-energy intermediates (adenosine triphosphate, phosphocreatine and pyridine nucleotides) were much higher in the “O” hr samples of “slow-glycolyzing” muscles than in similar samples from muscles having “fast” rates of post-mortem glycolysis. No significant differences in levels of lactate or glucose were observed among these three groups in blood samples taken either at or 24 hr prior to the time of exsanguination.
TL;DR: The physiological role of the Cu++-fructose 1,6-diphosphate interrelation in the balance between glycolysis and gluconeogenesis is discussed and the activation of the enzyme with NH4+ has been found to be more efficient than with K+.
TL;DR: A revision of the PK assay technique is suggested, since catalytic inefficiency of PK(2) was manifested only at low substrate concentrations and was therefore undetectable at the relatively high phosphoenolpyruvate levels employed in the conventional assay.
Abstract: Atypical cases of heritable hemolytic anemia have been noted that conform clinically and biochemically to anemias of the pyruvatekinase (PK)-deficient type, except for the presence of apparently adequate quantities of erythrocyte-PK activity by the usual assay procedure. Investigations of four such anomalous cases, occurring in two unrelated families, are presented. Erythrocytes contained a kinetically aberrant isozyme of pyruvate kinase (PK2). Michaelis constants for the pathologic isozyme relative to phosphoenolpyruvate were over 10-fold greater than control values, but no kinetic abnormality was evident for the second substrate, adenosine diphosphate. PK2 exhibited a pH optimum almost 1 U lower than the wild enzyme form (PK1). Significant differences were also evident in the functional stabilities of the isozymes. Leukocytes were unaffected.
Family studies revealed paternal heterozygosity for quantitative PK deficiency of the usual type. Clinically normal maternal relatives and some siblings demonstrated intermediate deviations in erythrocyte-PK kinetics and reaction characteristics compatible with coexistence of normal PK1 and kinetically abnormal PK2. Hemolytic anemia in the propositi appeared to require simultaneous inheritance of the gene governing PK2 production and its presumed allele resulting in quantitative PK deficiency. Both genetic defects were traced through three generations, the defective gene in both instances apparently resident on autosomes.
A revision of the PK assay technique is suggested, since catalytic inefficiency of PK2 was manifested only at low substrate concentrations and was therefore undetectable at the relatively high phosphoenolpyruvate levels employed in the conventional assay.
TL;DR: Phosphofructokinase appears to be a rate-limiting step in that part of the Embden-Meyerhof pathway which is present and the possible fate of phosphoenolpyruvates in relation to the apparent absence of pyruvate kinase is discussed.
TL;DR: Microcysts metabolized acetate at a lower rate than did vegetative cells and showed a different distribution of (14)C-label from incorporated acetate, reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2).
Abstract: Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO2. In addition, microcysts and vegetative cells showed a different distribution of 14C-label from incorporated acetate.
Journal Article•
TL;DR: These findings provide evidence for the suggestion offered many years ago by Johnson and by Lynen that the Pasteur effect may reflect competition for ADP and inorganic phosphate at the transphosphorylating sites of glycolysis and respiration.
Abstract: Summary In a continuation of studies on glycolytic and respiratory interrelationships in a series of rat hepatomas ranging widely in growth rate and degree of differentiation, whole, fortified homogenates of these tissues were incubated at 28°C in the presence of fructose-1,6-diphosphate (FDP), 2-deoxyglucose (2-DG), and exogenous hexokinase; respiration, lactate formation, and uptake of 2-DG were measured. Preliminary studies with this model system established that uptake of 2-DG was a valid measure of ATP formation. On the assumption that each mole of lactate formed from FDP leads to production of 2 moles of ATP via phosphoglycerate kinase and pyruvate kinase, glycolytic phosphorylation was estimated as twice that of lactate, and respiratory phosphorylation was calculated as the difference between total ATP and glycolytic ATP formation. Without exogenous substrate, respiration was high in homogenates of well-differentiated tumors, and low in those of poorly differentiated tumors. In both tumor types respiration was coupled with ATP formation, yielding P/O ratios of 1 to 2. Addition of FDP to liver homogenates resulted in moderate lactate and glycolytic ATP formation, the latter being formed largely at the expense of respiratory phosphorylation. In homogenates of well-differentiated tumors, lactate formation and glycolytic phosphorylation were low, and neither respiration nor respiratory phosphorylation was decreased. In contrast, homogenates of the poorly differentiated hepatomas exhibited high lactate formation, and though respiration was increased somewhat by FDP addition, essentially all of the ATP was formed via glycolysis. It thus appears that, in this system, transphosphorylating enzymes of glycolysis are a major site of glycolytic control, presumably through competition with the respiratory ADP acceptors for the available ADP. Further evidence for such competition was obtained by intermixing the supernatant and particulate fractions. Replacement of particles from a low-respiring, poorly differentiated tumor by particles from a high-respiring, well-differentiated tumor resulted in a pronounced Pasteur effect; respiration was increased, together with respiratory ATP production, while glycolysis was markedly decreased. However, when particles of a high-respiring tumor were replaced with particles of a low-respiring tumor, respiration and respiratory phosphorylation were decreased and glycolysis was markedly increased. These findings provide evidence for the suggestion offered many years ago by Johnson and by Lynen that the Pasteur effect may reflect competition for ADP and inorganic phosphate (P 1 ) at the transphosphorylating sites of glycolysis and respiration. They also suggest that the high aerobic glycolysis which is, in general, characteristic of highly dedifferentiated tumors may be, in part, a resultant of their low respiratory activity and high levels of glycolytic transphosphorylating enzymes.
TL;DR: FFA which is an end product of the degradation of FFA reconfirms the feedback inhibition by inhibiting glucokinase and pyruvate kinase activities and l -alanine acted as a competitive inhibitor against PEP.
TL;DR: It is suggested that pyruvate can be formed from phosphoenolpyruVate by a pathway involving the action of phosphoenlpyruvates carboxylase, malate dehydrogenase, and malate dehydration enzyme, as well as the significance of such a pathway is discussed.
TL;DR: Assays of the activities of the soluble and the mitochondrial pyruvate carboxylase after administration of cortisol support a regulation of gluconeogenesis by control of the extramitochondrial enzyme levels, and alanine proved to be a potent inhibitor of the enzyme from liver.
TL;DR: The results show that the PK-deficient cell is heterogeneously affected by whatever mechanisms induce its destruction, and a population of "young" PK cells contains more seriously damaged cells than apopulation of older cells, the latter living to an older age because of their improved initial outlook.
Abstract: To define the role of the spleen in the hemolytic anemia associated with erythrocyte pyruvate kinase (PK) deficiency, removal rates and patterns or organ sequestration of 51Cr-labeled PK erythrocytes were studied. Recipients were the PK patients themselves, normal subjects and persons splenectomized for diverse causes. In one study the 51Cr was attached to "young" and "old" populations of PK erythrocytes before their injection; in another the "young" population was differentially labeled with glycine-2–14C. The results show that the PK-deficient cell is heterogeneously affected by whatever mechanisms induce its destruction. Therefore, a population of "young" PK cells contains more seriously damaged cells than a population of older cells, the latter living to an older age because of their improved initial outlook. Younger cells, if they pass through the spleen, are subsequently destroyed in the liver. Removal of the spleen improves the survival of the younger cells, for they are then destroyed muc...
TL;DR: The findings are against an oxygen deficit as a cause of cardiac deterioration in shock, suggesting structural derangements possibly due to catecholamine effects or toxic substances of peripheral origin.
Abstract: Twelve experiments were performed on healthy, anesthetized dogs on constant ventilation. Blood pressure, acid-base status, oxygen supply and lactate/pyruvate metabolism were monitored in the blood. The activities of nine glycolytic enzymes were assayed in left ventricular myocardium. A presumably irreversible state of hemorrhagic shock was produced by the reservoir technique and its metabolic sequels recorded in the blood. The analysis of coronary sinus blood did not reveal an oxygen deficit of the heart. The majority of the enzymes assayed showed higher activities following shock. Exceptions were pyruvate kinase, LDH and α-HB-DH. Activity differences between sampling sites appeared following shock especially in the inner layer of the left ventricle. The pattern of transmural activity gradients previously found in the normal state was disturbed. The findings are against an oxygen deficit as a cause of cardiac deterioration in shock, suggesting structural derangements possibly due to catecholamine effects or toxic substances of peripheral origin.
TL;DR: Liver from a patient with hereditary haemolytic anaemia due to pyruvate kinase deficiency is a deficient in the ‘erythrocyte’ PK isozyme as are her ery Throcytes, and the chromatographic, electrophoretic, antigenic, heat stability and kinetic properties of the patient's enzyme were not different from normal.
Abstract: SUMMARY
Liver from a patient with hereditary haemolytic anaemia due to pyruvate kinase deficiency is a deficient in the ‘erythrocyte’ PK isozyme as are her erythrocytes. The chromatographic, electrophoretic, antigenic, heat stability and kinetic properties of the patient's enzyme were not different from normal.
TL;DR: It is concluded that any great acceleration in the rate of gluconeogenesis via glycolysis during culmination is brought about by changing substrate levels or by intracellular relocalization rather than by an increase in total enzyme activity.
TL;DR: B Bidirectional effects on the velocity of human PK I reactions, which depend upon the concentration of FDP, are described and a model to account for these observations is proposed.
Journal Article•
TL;DR: It is suggested that ATP and ATPase may participate in the release of hormones from the intact posterior pituitary gland.
Abstract: Neurosecretory granules were isolated from bovine posterior pituitary glands and incubated at 30 or 37° in a medium containing KCl and MgCl2. On exposure to ATP the granules released vasopressin, oxytocin, and protein. Vasopressin release in response to ATP was potentiated by phosphoenolpyruvate + pyruvate kinase, was inhibited by AMP, and was accompanied by a fall in the optical density (at 540 mµ) of the granule suspension. The neurosecretory granules possessed ATPase activity and ATP. It is suggested that ATP and ATPase may participate in the release of hormones from the intact posterior pituitary gland.
TL;DR: Total and specific hexokinase activities decreased significantly in homogenates and particle-free supernatants prepared from the intestinal mucosa of rats starved for 36hr and increased again after re-feeding.
Abstract: 1. The intracellular distribution of hexokinase activity was studied in the mucosa of rat and guinea-pig small intestine. In the rat 60% and in the guinea pig 45% of the hexokinase activity of homogenates were recovered in a total particulate fraction that contained only 5–17% of the homogenate activity of hexose phosphate isomerase, pyruvate kinase, lactate dehydrogenase and overall glycolysis (formation of lactate from glucose). 2. Fractionation of homogenates from guineapig small intestine showed that the particulate hexokinase activity was chiefly in the mitochondrial fraction with a small proportion in the nuclei plus brush-border fraction. 3. After chromatography of the particle-free supernatants on DEAE-cellulose, hexokinase types I and II were determined quantitatively. No evidence was obtained for the presence of hexokinase type III or glucokinase. In the preparations from guinea pigs, hexokinase types I and II amounted to 69% and 31% respectively of the eluted activity; the corresponding values for preparations from rats were 5·8% and 94·2%. 4. Total and specific hexokinase activities decreased significantly in homogenates and particle-free supernatants prepared from the intestinal mucosa of rats starved for 36hr. and increased again after re-feeding. The decrease in hexokinase activity in the particle-free supernatant from starved rats was chiefly due to a decrease in the type II enzyme.
TL;DR: The covalent attachment of pyruvate kinase to filter-paper disks and some kinetic properties of the resultant enzymically active porous sheets are described.
Abstract: 1. The covalent attachment of pyruvate kinase to filter-paper disks and some kinetic properties of the resultant enzymically active porous sheets are described. 2. A two-enzyme reactor was constructed by using water-insoluble sheets of lactate dehydrogenease in conjunction with the above-mentioned sheets possessing pyruvate kinase activity.