Showing papers on "Pyruvate kinase published in 1976"

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

Abstract: The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.

Hormonal control of pyruvate kinase activity and of gluconeogenesis in isolated hepatocytes.

Abstract: Treatment of isolated rat hepatocytes with saturating concentrations of glucagon caused several modifications properties of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40): S0.5 (substrate concentration at half maximum velocity) for phosphoenolpyruvate was about doubled, whereas Vmax was not changed; the activity measured at 0.15 mM phosphoenolpyruvate (physiological concentration) was reduced 65-80%; and there was also an increase in the Hill coefficient and in the affinity of the enzyme for the inhibitors Mg-ATP and alanine. Glucagon, 3':5'-cyclic AMP, and epinephrine caused an inactivation of pyruvate kinase together with a sitmulation of gluconeogenesis. Insulin (10 nM) antagonized the effect of suboptimal doses of glucagon or cyclic AMP and of even maximal doses of epinephrine, on both pyruvate kinase activity and on gluconeogenesis. These observations can be explained by a phosphorylation of pyruvate kinase by cyclic-AMP-dependent protein kinase, as described by Ljungstrom et al. [(1974) Biochim. Biophys. Acta 358, 289-298] in a reconstructed system. They offer a molecular explanation for the hormonal control of gluconeogenesis. Glucose caused an inhibition of gluconeogenesis with no corresponding change in pyruvate kinase activity.

Liver Cell Heterogeneity. The Distribution of Pyruvate Kinase and Phosphoenolpyruvate Carboxykinase (GTP) in the Liver Lobule of Fed and Starved Rats

Abstract: Pyruvate kinase and phosphoenolpyruvate carboxykinase activities were determined in microdissected freeze-dried liver cells from the periportal and pericentral area of the liver lobule. Pyruvate kinase activity was measured by a microfluorimetric procedure adapted to 20-200 ng tissue dry weight. In livers from fed rats, its activity was twice as high in the central zone as in the periportal cells; starvation reduced this gradient by decreasing central activities. Phosphoenolpyruvate carboxykinase activity was measured by a microradiochemical technique in 100-300 ng tissue dry weight. In livers from fed rats, this enzyme was nearly 3 times more active in the periportal cells than in the central area. Starvation increased this enzyme in both zones with a more pronounced change in the central cells. The results indicate a heterogeneous distribution of enzymes of carbohydrate metabolism in the liver lobule. Gluconegenesis seems to be localized preferentially in periportal hepatocytes, whereas the glycolytic enzyme was found to be more active in cells surrounding the pericentral liver cells.

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Abstract: Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.

Changes in mitochondrial phosphorylative activity and adenylate energy charge of regenerating rabbit liver.

Abstract: The changes in the cellular concentrations of ATP, ADP, and AMP and in oxidative phosphorylation of mitochondria were investigated in the remaining liver of partially hepatectomized rabbits. The energy charge (defined as half of the average number of anhydride-bonded phosphate groups per adenosine moiety) of the liver remnant decreased from 0.866 to 0.767 (p less than 0.01) within 24 hr after hepatectomy, and then increased to a substantially higher level than normal within 7 days. On the other hand, the mitochondrial phosphyorylative activity increased rapidly to 170 per cent of the control within 12 hr and then retruned to normal within 7 days. The mitochondrial phosphorylative activity was inversely correlated with energy charge of the liver remnant (r = -0.75, p less less than 0.01). The maximal enhancement of mitochondrial phosphorylative activity was found in mitochondria obtained from the liver remnant with the lowest level of energy charge, suggesting a response of mitochondria in vivo involving enhanced biosynthetic ATP-utilizing reactions at an early stage of the regenerating process. The enhancement of phosphorylative activity was accompanied by a rise in the respiratory control ratio, P/O ratio and state 3 respiration. The adenylate kinase [EC 2.7.4.3] activity in the liver remnant increased to more than 160% of the control within 2 days after partial hepatectomy, while the pyruvate kinase [EC 2.7.1.40] activity decreased remarkably. However, the changes in the two enzyme activities did not correlate with those of mitochondrial phosphorylative activity or the energy charge of the liver remnant.

Inhibition of acetylcholine synthesis and of carbohydrate utilization by maple-syrup-urine disease metabolites.

Abstract: The branched-chain 2-oxo acids which accumulate in maple-syrup-urine disease inhibited the production of acetylcholine and of lipids, proteins, nucleic acids and of CO2. in sliced adult rat brains incubated with [U-14C] glucose. Inhibition of the biosynthetic reactions was proportional to the inhibition of CO2 production, even though the flux of radioactivity into the biosynthetic products was less than 2% of that to CO2. The oxo acids reduced the production of 14CO2, from [U-14C] glucose and from [2-14C]pyruvic acid more than from [1-14C]pyruvic acid in sliced brains. They inhibited the solubilized oxoglutarate dehydrogenase complex more than they did the solubilized pyruvate dehydrogenase complex. Valine and isoleucine, which also accumulate in maple-syrup-urine disease, inhibited pyruvate kinase from rat brain allosterically. Quantitative comparison of the effects of the disease metabolites on cell-free systems with their effects on fluxes in intact cells indicated that the inhibition of oxoglutarate dehydrogenase appeared to be functionally significant. The residual activities of the other enzymes studied were adequate to support the normal flux of carbohydrates. The oxo acids were effective at concentrations within the range reported to occur in patients with maple-syrup-urine disease. The effects on biosyntheses including that of acetylcholine would be expected to impair brain development and function and could be important in the development of brain disease in the patients. In contrast to the results with metabolites from maple-syrup-urine disease, metabolites which accumulate in phenylketonuria (phenylalanine and 2-oxo-3-phenylpropionic acid) did not inhibit carbohydrate utilization or the biosynthetic reactions studied, under the conditions of these experiments.

Regulation of lactose fermentation in group N streptococci.

Abstract: Group N streptococci, which have the lactose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) and phospho-β-d-galactosidase (β-Pgal), grew rapidly on lactose and converted more than 90% of the sugar to l-lactate. In contrast, Streptococcus lactis 7962, which does not have a β-Pgal, grew slowly on lactose and converted only 15% of the sugar to l-lactate. With glucose and galactose, this strain had growth rates and fermentation patterns similar to those of other S. lactis strains, suggesting that the rapid and homolactic fermentation of lactose that is characteristic of group N streptococci is dependent upon a functional PEP-dependent PTS and the presence of β-Pgal. Seventeen strains of group N streptococci were examined for the activator specificities of pyruvate kinase and lactate dehydrogenase. The properties of each enzyme from all the strains, including S. lactis 7962, were similar. Pyruvate kinase had a broad activator specificity, whereas activation of lactate dehydrogenase was specific for ketohexose diphosphate. All intermediates of lactose metabolism from the hexose phosphates to the triose phosphates activated pyruvate kinase. No activation was obtained with adenosine 5′-monophosphate. K+ and Mg2+ were required for pyruvate kinase activity but could be replaced by NH4+ and Mn2+, respectively. Lactate dehydrogenase was activated equally by fructose-1,6-diphosphate and tagatose-1,6-diphosphate, the activation characteristics being pH dependent. The roles of pyruvate kinase and lactate dehydrogenase in the regulation of lactose fermentation by group N streptococci are discussed.

Effects of Thyroid Hormone Deficiency on the Distribution of Hepatic Metabolites and Control of Pathways of Carbohydrate Metabolism in Liver and Adipose Tissue of the Rat

Abstract: 1 Measurements have been made of the hepatic metabolites in normal and thyroidectomized rats and of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and of lipogenesis together with the flux of glucose through alternative pathways of glucose metabolism and into lipid. 2 There is a significant fall in the content of ADP, AMP, citrate, long-chain acyl-CoA derivatives and a rise of 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate following thyroidectomy. The observed changes in the glycolytic intermediates may be correlated with the increased activity of phosphofructokinase relative to pyruvate kinase and fructose bisphosphatase. 3 The NAD+/NADH quotient of the mitochondrial compartment, calculated from the reactants and Keq of 3-hydroxybutyrate dehydrogenase, becomes significantly more oxidized in hypothyroid animals. The redox state of the cytosolic NAD and NADP couples remains relatively unchanged. 4 The changes in the hepatic content of CoA derivatives and citrate and in the mitochondrial redox state are interpreted as indicating a depressed rate of lipid oxidation. 5 Calculations of the compartmentation of metabolites between the cytosol and mitochondria indicate a very marked decrease in mitochondrial citrate, 2-oxoglutarate and glutamate with smaller changes in aspartate and malate. These changes are interpreted as providing evidence for the importance of modifications in the malate-aspartate shuttle in hypothyroidism; this is further supported by measured changes in the distribution and activities of the component enzymes of the hydrogen shuttles. 6 There is a diminished activity of glucokinase and of enzymes of the glycolytic pathway below phosphofructokinase in livers from hypothyroid rats. The oxidative enzymes of the pentose phosphate pathway, ATP-citrate lyase, ‘malic’ enzyme and fatty acid synthetase also decrease markedly. There is a striking parallelism between the changes of enzyme profile of liver and adipose tissue in hypothyroidism. 7 Adrenal glands from hypothyroid rats showed a generalized decline in enzyme activity in parallel with the fall in tissue weight. Cytosolic glycerol-3-phosphate dehydrogenase decreased sharply. 8 The present results are discussed in relation to data in the literature on the increased activities of cyclic AMP and cyclic GMP phosphodiesterases in hypothyroidism, observations which integrate the relationships between anabolic and catabolic pathways of carbohydrate and lipid metabolism with alterations in hormone sensitivity in hypothyroidism.

The control of pyruvate kinase of Escherichia coli. Binding of substrate and allosteric effectors to the enzyme activated by fructose 1,6-bisphosphate.

Abstract: The binding of various regulatory ligands and substrates to the fructose bisphosphate activated pyruvate kinase from Escherichia coli has been studied at equilibrium The allosteric activator, fructose bisphosphate, and the substrate phosphoenolypyruvate bind in a cooperative manner to the enzyme There is one site for each of these ligands per monomer In the presence of fructose bisphosphate the binding of phosphoenolpyruvate follows an absorption isotherm, ie, all homotropic interactions of the substrate are lost In reciprocal experiments, however, both phosphoenolpyruvate and KCl are required in order to facilitate binding of the activator The allosteric inhibitors of pyruvate kinase, ATP, succinyl-CoA, and GTP compete on the enzyme surface with the binding of the activator, fructose bisphosphate, Inhibitor pairs such as ATP and succinyl-CoA together bring about not cooperative but only additive inhibition of the binding of the activator The nucleotide substrate GDP and the allosteric inhibitor GTP have in contrast to the activator two seemingly noninteracting sites on each monomer In the saturating presence of fructose bisphosphate, however, binding of GDP and possibly also of GTP occurs at only one site on each monomer Magnesium ions inhibit binding of GDP and GTP KCl which is an activator of the enzyme along with its analogues, such as ammonia, thallium, rubidium, etc, enhances the binding of phosphoenolpyruvate but not of the nucleotides or fructose bisphosphate The data are analyzed on the basis of a two-site model, where the substrate and fructose bisphosphate bind to one conformation and the inhibitors to the other

Studies on the Kinetic Effects of Adenosine‐3′: 5′‐MonophosphateDependent Phosphorylation of Purified Pig‐Liver Pyruvate Kinase Type L

Abstract: The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, Ko.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-bisphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-bisphospliateactivated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered-from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.

Pyruvate kinase isozymes in neurons, glia, neuroblastoma, and glioblastoma.

Abstract: – The distribution of pyruvate kinase isozymes (EC 2.7.1.40) was examined in cells and tissues from the central and peripheral nervous system of the rat. Most tissues contain significant quantities of both the K4 (fetal type) and M4 (skeletal muscle type) isozymes plus tetrameric hybrids comprised of various combination of the type M and type K subunits. Retina, for example, contains a five-mem-bered hybrid set weighted toward K4, while sciatic nerve and spinal cord have patterns very similar to that of adult brain, consisting predominantly of M4 with small amounts of K4 and K-M hybrids. This adult pattern is achieved by a gradual shift from a hybrid set dominated by K4 in fetal life, to the pattern at birth at which time the two most prominent bands were M4 and K2M2, and finally to the adult pattern by about 28 days after birth. Neurons and glial cells were isolated from rat and mouse brains at the various developmental levels. The pyruvate kinase isozyme patterns in the two cell types were similar to each other and to the patterns seen in whole brain homogenates at all ages, indicating similar rates of isozymic maturation in the two cell types. The correlation of maturation with pyruvate kinase isozyme patterns was further tested in cultures of malignant cell lines. A K-M hybrid set, weighted toward K4, was seen in two clonal lines of mouse neuroblastoma under normal culture conditions. However, lowering the serum concentration in the culture medium or adding bromodeoxyuridine caused a shift in the patterns toward type M as the cells differentiated, mimicking in part the in vivo maturation of normal cells. On the other hand, a rapidly growing and poorly differentiated line of rat glioblastoma had only K4 under all conditions examined.

Pyruvate kinase isozymes in man. I. M type isozymes in adult and foetal tissues, electrofocusing and immunological studies.

Abstract: Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not at all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes.

Rapid Metabolic Changes in the Wounding Response of Leaf Discs following Excision.

Abstract: The dark respiration rate of discs from fully expanded tobacco leaves (Nicotiana tabacum) increased linearly with decreasing diameter, the relative increase being independent of leaf age The wound respiration responsible for this situation reached a plateau within 15 minutes of excision Metabolite analysis gave evidence for two independent effects, also unrelated to age The first was a forward crossover between phosphoenolpyruvate and pyruvate which was found as early as 1 minute after excision and persisted for up to 40 minutes It was attributed to activation of pyruvate kinase by a changed ionic balance resulting from membrane damage, was accompanied by a reverse crossover between triose phosphates and 3-phosphoglycerate, and was localized in the outer region of the discs The second effect was a rapid rise in hexose monophosphate and ATP levels throughout the discs After 1 to 10 minutes the ATP/ADP ratio rose strongly for at least 3 hours; after 20 to 40 minutes there was net synthesis of adenine nucleotide as ATP These results indicate that extrapolation from leaf discs to intact leaves is highly inadvisable

Inhibition of chicken pyruvate kinases by amino acids.

Abstract: Alanine, serine, and phenylalanine behave as inhibitors competitive with phosphoenolpyruvate for the activated forms of the chicken pyruvate kinases. On the other hand, phenylalanine and alanine behave as K-type inhibitors and serine behaves as a heterotropic activator of pyruvate kinase variants which undergo homotropic activation. Tryptophan lowers the Vm and tends to yield complex plots with all variants studied. Kinetic patterns obtained in the presence of phenylalanine also show some characteristics not generally associated with a competitive mechanism. These observations are related to data previously obtained using the rat isozymes and are used to formulate a mechanism which explains the effects of the amino acids. This mechanism hypothesizes that all the effector amino acids bind to the phosphoenolpyruvate site; however, amino acids with nonpolar side chains also interact with a nonpolar region of the T conformer and thereby stabilize it. It is further proposed that there are two such nonpolar regions on the various pyruvate kinases--the one which reacts with the nonbulky side chains, and another which reacts only with relatively bulky side chains. The stabilizing effect of this second nonpolar interaction imparts inhibitory characteristics which are not competitive in nature. Serine and perhaps other polar compounds may also bind at the phosphoenolpyruvate site, but because of their polarity exert a repulsive force at the same nonpolar site with which the nonbulky nonpolar amino acids interact. This repulsion stabilizes the R conformation. Presumably the homotropic activating effects of phosphoenolpyruvate operate via this same mechanism. The data are also used to support a specific sequential-concerted mechanism for the homotropic activating effect of phosphoenolpyruvate. According to this mechanism, phosphoenolpyruvate adds sequentially to the first two subunits. This interaction causes the respective subunits to convert to the R conformation but, once two subunits are in R conformation, the remaining two subunits convert in concert.

Activator Specificity of Pyruvate Kinase from Lactic Streptococci

Abstract: The pyruvate kinase from lactic streptococci was activated by 20 structurally dissimilar sugar phosphates and glycolytic intermediates. Nine compounds were more effective activators than fructose-1,6-diphosphate.