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PubMed1983

Distribution of pyruvate kinase type L and M2 in microdissected periportal and perivenous rat liver tissue with different dietary states.

Pyruvate kinase type L and M2 activities were measured in microdissected periportal and perivenous liver tissue from rats in different dietary states. A specific antibody against pyruvate kinase type L was used to distinguish the two isoenzymes. Using separated cells it was found that the L-isoenzyme was essentially restricted to the parenchymal and the M2-isoenzyme to the non-parenchymal cells. Pyruvate kinase type L activity in the perivenous zone was about twice as high as in the periportal zone in both male and female fed rats. Starvation for 48 h led to a decrease of the overall activity and to a lower perivenous-periportal gradient. After refeeding for 48 h the overall activity and the gradient were increased to above the normal level. Pyruvate kinase type M2 was homogenously distributed within the liver acinus. After 48 h starvation no change in the overall activity nor in the zonal distribution was observed. Refed rats exhibited a slightly reduced overall activity. Since the hepatocytes contain the total regulatory L- but no M2-pyruvate kinase the heterogeneous distribution of the L-isoenzyme under different dietary states supports the model of metabolic zonation of liver parenchyma with glycolysis predominantly in the perivenous zone.

Animals↗
PubMed2000

Purification and characterization of cytosolic pyruvate kinase from banana fruit.

Cytosolic pyruvate kinase (PK(c)) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7 micromol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240 kDa homotetramer composed of subunits of 57 kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of V(max)/K(m) for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH 6.9 and 7.5. PK(c) activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg(2+) and K(+) respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg(2+) and K(+) (K(m) values of 0.098, 0.12, 0.27 and 0.91 mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PK(c) by L-glutamate. The allosteric features of banana PK(c) are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807-816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

Adenosine Diphosphate↗
PubMed1979

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.

Adolescent↗
PubMed1993

The pyruvate kinase gene as a model for studies of glucose-dependent regulation of gene expression in the endocrine pancreatic beta-cell type.

The insulinoma beta-cell line INS-1 expresses the L-type pyruvate kinase gene at high level and responds to a rise in extracellular glucose by strong induction of gene expression. Following the addition of glucose to the culture medium in the 3.5-33 mM concentration range, the cellular level of L-type pyruvate kinase mRNA increases within 2 h and reaches a maximum 15-fold above basal in 8-12 h. By run-on nuclear assay, the relative transcription rate of the pyruvate kinase gene is shown to increase 4-fold at maximal stimulation, suggesting that both transcriptional and post-transcriptional effects contribute to mRNA accumulation. The glucose effect is totally suppressed by the hexokinase inhibitor mannoheptulose, indicating a requirement for glucose phosphorylation. The mRNA induction is not inhibited in glutamine-free culture medium or by azaserine, suggesting that the hexosamine biosynthetic pathway is not involved. Moreover, metabolism along the glycolytic pathway does not appear to be an absolute requisite, since 2-deoxyglucose partly mimics the inductive effect of glucose. The glucose effect on the pyruvate kinase gene is reversibly antagonized by agents increasing intracellular cAMP. In addition, the effect is highly specific to the pyruvate kinase gene. Neither proinsulin I mRNA nor glucokinase mRNA are increased in glucose-stimulated INS-1 cells. Short term transfection with CAT plasmids driven by the pyruvate kinase L promoter reveals specific glucose-inducible reporter activity with the 183-base pair promoter region upstream of the cap site. Within this region, the previously described L4 cis-acting element is crucial for glucose responsiveness, as demonstrated by the fact that a plasmid with a mutation in this element does not elicit glucose-inducible CAT activity. Induction of L-type pyruvate kinase mRNA occurs in the islets of rats subjected to fasting and carbohydrate refeeding. In conclusion, the L-type pyruvate kinase gene provides an interesting model of glucose-regulated gene in the endocrine beta-cell type.

Animals↗
PubMed1975

Pyruvate kinase isozymes in adult and fetal tissues of chicken.

Tissues of fetal and adult chickens were examined for pyruvate kinase activity. Two electrophoretically distinguishable and noninterconvertible isozymes were found. One of these, designated as type K (for kidney), is the sole pyruvate kinase in the early fetus and is found in appreciable quantities in all adult tissues except striated muscle. The second isozyme, type M, appears shortly before hatching in striated muscle and brain. These two isozymes correspond in their developmental pattern, tissue distribution, electrophoretic, immunological, and kinetic propertiesto similarly designated mammalian pyruvate kinases. However, no kinetic, immunological, or electrophoretic evidence could be found for a chicken isozyme corresponding to the mammalian type L pyruvate kinase. As the latter isozyme seems to be limited in its distribution mostly to highly differentiated gluconeogenic tissues (notable liver, kidney, and small intestine), our results support the proposition that the mammalian type L pyruvate kinase is a specilized isozyme that is present in mammals but not in birds.

Aging↗
PubMed1979

Effect of endotoxin on hepatic pyruvate kinase activity in normal and diabetic dogs.

The influence of endotoxic administration (0.5 mg/kg) on hepatic pyruvate kinase activity in normal (nondiabetic) and diabetic dogs was investigated. Pyruvate kinase activity was not affected four hours after endotoxin injection in nondiabetic dogs. However, it was stimulated by 288% in dogs that were made diabetic prior to the administration of endotoxin. Since pyruvate kinase activity in diabetic dogs without endotoxin treatment was not affected, these findings suggest that the stimulatory effect of endotoxin on pyruvate kinase activity in diabetic dogs may be associated with insulin deficiency and/or high ambient blood glucose level.

Animals↗
PubMed1999

Negative cyclic AMP response elements in the promoter of the L-type pyruvate kinase gene.

L-type pyruvate kinase gene expression is modulated by hormonal and nutritional conditions. Here, we show by transient transfections in hepatocytes in primary culture that both the glucose response element and the contiguous hepatocyte nuclear factor 4 (HNF4) binding site (L3) of the promoter were negative cyclic AMP (cAMP) response elements and that cAMP-dependent inhibition through L3 requires HNF4 binding. Another HNF4 binding site-dependent construct was also inhibited by cAMP. However, HNF4 mutants whose putative PKA-dependent phosphorylation sites have been mutated still conferred cAMP-sensitive transactivation of a L3-dependent reporter gene. Overexpression of the CREB binding protein (CBP) increased the HNF4-dependent transactivation but this effect remained sensitive to cAMP inhibition.

Animals↗
PubMed1989

Independent regulation of pyruvate kinase expression by cyclic AMP and prostaglandin F2 alpha in mouse mastocytoma cells.

P-815 mouse mastocytoma cells express the K isozyme of pyruvate kinase and the specific activity of this enzyme is increased in response to N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, 8-bromoadenosine 3':5'-cyclic monophosphate, cholera toxin, and epinephrine, all of which also elevate the intracellular concentration of adenosine 3':5'-cyclic monophosphate. Prostaglandin F2 alpha also increases the cellular activity of this enzyme, but does not increase the adenosine 3':5'-cyclic monophosphate levels. Under all these conditions, the increase in enzymatic activity is accompanied by an equivalent increase in the pyruvate kinase protein level. However, neither the rate of enzyme synthesis nor the level of pyruvate kinase mRNA is elevated by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. On the other hand, it does increase the enzyme's half-life. In contrast, prostaglandin F2 alpha increases the rate of synthesis and the level of pyruvate kinase K mRNA, but has no influence on the rate of degradation. Therefore, these cells have two mechanisms which increase pyruvate kinase K levels. One operates via an increase in cAMP level and results in a decrease in the rate of degradation, whereas the other minimizes an upsurge in cAMP levels but still increases pyruvate kinase K activity by increasing its rate of synthesis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Activation of Fc epsilon RI inhibits the pyruvate kinase through direct interaction with the gamma-chain.

The downstream signaling components of high-affinity IgE receptor (FcepsilonRI) were studied using yeast two-hybrid screening of the cDNA library constructed from RBL-2H3 cells. The cytoplasmic part of the gamma-chain but not that of the beta-chain was found to interact with pyruvate kinase in the yeast. The in-vitro-translated pyruvate kinase also specifically interacted with the bacterially expressed glutathione-S transferase fusion protein of the cytoplasmic part of the gamma-chain. When RBL-2H3 cells were challenged with antigen, the activity of pyruvate kinase gradually decreased, reaching the minimum activity around 5 min after the activation, and then slowly returned to the normal level. The dose-response curve (antigen vs. pyruvate kinase activity) plotted at 5 min after stimulation showed that the pyruvate kinase was dose-dependently inhibited and the maximum inhibition was reached at the concentration of 0.1 microgram/ml of antigen. Direct interaction between FcepsilonRI and pyruvate kinase was also demonstrated by co-immunoprecipitation in RBL-2H3 cells. These data suggest that pyruvate kinase is functionally linked with FcepsilonRI and might exert an important role in controlling cellular functions following the activation of FcepsilonRI.

Animals↗
PubMed2003

Characterization of the inhibition of pyruvate kinase caused by phenylalanine and phenylpyruvate in rat brain cortex.

Pyruvate kinase plays a crucial role on the glycolytic pathway, the main route that provides energy for brain functioning. In the present study, we investigated the kinetics of the inhibition of pyruvate kinase provoked by phenylalanine and its main metabolite, phenylpyruvate, in mitochondria-free cerebral cortex homogenate from 22-day-old Wistar rats. We found that phenylalanine and phenylpyruvate inhibit PK activity by competition with the enzyme substrates ADP and phosphoenolpyruvate. We also investigated the interaction between phenylalanine and phenylpyruvate, and the kinetics of alanine prevention of the inhibitory action of phenylalanine and phenylpyruvate on pyruvate kinase activity. We observed that alanine per se had no effect on PK activity but prevented the inhibitory action of phenylalanine and phenylpyruvate by competition. The data suggest that phenylalanine, phenylpyruvate, and alanine act on a common site in the enzyme, probably an allosteric one. It is possible that inhibition of brain PK activity may be related to the reduction of glucose metabolism observed in the brain of phenylketonuric patients and may be one of the mechanisms responsible for the neurological dysfunction found in these patients. Further studies, however, are necessary to evaluate the benefit of carbohydrate and alanine supplementation to the diet of phenylketonuric patients.

Adenosine Diphosphate↗
PubMed1984

Regulation of glycolysis in lizards: kinetic studies on liver pyruvate kinase and phosphofructokinase from Lacerta galloti.

Kinetic studies were carried out on pyruvate kinase and phosphofructokinase from the lizard Lacerta galloti. Pyruvate kinase is inhibited by ATP and activated by fructose 1,6-biphosphate giving an hyperbolic saturation curve for ATP without the activator which becomes sigmoidal at saturating concentrations of fructose 1,6-biphosphate, giving a moderate cooperativity with a Hill coefficient of h = 1.72. Binding of fructose 1,6-biphosphate to pyruvate kinase was studied as protection effect against thermal denaturation, this being the most suitable ligand tested to avoid the loss of activity. Phosphofructokinase is inhibited by ATP at millimolar range and activated by AMP and by fructose 2,6-biphosphate, AMP being the more efficient activator.

Adenosine Monophosphate↗
PubMed1982

Kinetic properties of human muscle pyruvate kinase.

The steady-state kinetics of human skeletal muscle pyruvate kinase (MA) and its RNA-complex (MB) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase. The MA form follows a simple Michaelis-Menten kinetics in contrast with the MB form, which displays a negative cooperativity with respect to ADP. Vmax for the complex is 40-60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (MB) is not affected. In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for Vmax, which increased by nearly 60%. Aged preparations of free enzyme (MA) were activated by 100% and more, but the native enzyme was inhibited by 22%. Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but Vmax for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged MA form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.

Enzyme Activation↗
PubMed1991

A rate-determining proton relay in the pyruvate kinase reaction.

This study ascribes the large steady-state D2O isotope effect on kcat of pyruvate kinase (PEP + ADP----pyruvate + ATP) to the reprotonation of the product form of the enzyme for use in forming pyruvate. Previous tritium trapping experiments [Rose, I. A., & Kuo, D. J. (1989) Biochemistry 28, 9579-9585] with muscle pyruvate kinase showed that the proton used for ketonization of enolpyruvate is derived from an enzyme "pool" that contains three kinetically equivalent hydrogens that could be trapped in a nontritiated "chase" medium by high levels of ADP and PEP. The exchange of this pool with the medium was rapid in the free enzyme (approximately 1400 s-1), prior to addition of PEP, and apparently much less in the completed complex. The dissociation rate constant was determined by using the dissociation-competition equation koffT = K1/2kcat/Km, where kcat/Km is the steady-state parameter for PEP and K1/2 is the concentration of PEP required to trap 50% of the isotope that could be trapped. The present study shows that the competition constant, K1/2, is decreased by approximately 5-fold in D2O, the same effect see on kcat under conditions where kcat/Km, measured in the steady state, is not changed. The common effect of D2O on kcat in the steady state and koffT in pulse/chase suggests that the forward reaction rate is determined by hydrogen transfer to the enzyme. Further evidence indicates that the kinetically important proton in question is the proton used for ketonization of enolpyruvate, the substrate proton.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Antagonizing effects of phorbol 12-myristate 13-acetate on hormonally stimulated gluconeogenesis in isolated rat hepatocytes involve activity changes of pyruvate kinase.

The tumor-promoting phorbol ester phorbol 12-myristate 13-acetate partially neutralized the stimulatory effects of epinephrine (alpha 1-adrenergic actions), glucagon, and dibutyryl-cAMP on gluconeogenesis in isolated hepatocytes of fasted rats, when lactate or dihydroxyacetone was used as the substrate. By constructing metabolic crossover plots and by comparing rates of lactate production from dihydroxyacetone with K0.5 values of extracted pyruvate kinase for phosphoenolpyruvate, we obtained evidence that phorbol ester actions on hormonally stimulated gluconeogenesis were accompanied by proportionate increases in activity of pyruvate kinase. Although purified pyruvate kinase from rat liver was a substrate for protein kinase C in vitro, phosphorylation was not accompanied by modulation of kinetic parameters. Furthermore, incubation of pyruvate kinase extracted from hormone-treated hepatocytes with protein kinase C revealed no activation of the prephosphorylated enzyme. This and the absence of effects of the phorbol ester on basal rates of gluconeogenesis and lactate production suggest that effects of protein kinase C on pyruvate kinase activity in hepatocytes may result from impairment of steps at the level of hormone-induced signal transduction.

Animals↗
PubMed1997

Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger.

Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3-5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate.

Aspergillus niger↗
PubMed1966

Studies on the interaction of metal ions with pyruvate kinase from Ehrlich ascites-tumour cells and from rabbit muscle.

1. Pyruvate kinase (ATP-pyruvate phosphotransferase, EC 2.7.1.40) from Ehrlich ascites-tumour cells was purified approximately fivefold by chromatography on DEAE-cellulose. The enzyme was shown to have an absolute requirement for one univalent and for one bivalent metal ion. 2. The univalent metal ion requirements were satisfied by K(+), Rb(+) or NH(4) (+); Na(+) and Cs(+) were weak activators but Li(+) was inactive. 3. Ca(2+) exhibited ;non-competitive' and ;apparent competitive' effects in relation to the K(+) activation. 4. The bivalent metal ion requirements were satisfied by Mg(2+), Mn(2+) or Co(2+); Ba(2+), Sr(2+), Ca(2+), Ni(2+), Be(2+) and Cu(2+) were inactive. Mn(2+) and Co(2+) were better activators than Mg(2+). 5. The bivalent metal ion requirements of purified pyruvate kinase from rabbit muscle were satisfied by Mg(2+), Mn(2+), Co(2+) and to a smaller extent by Ni(2+). Mn(2+) and Co(2+) were better activators than Mg(2+). 6. Ca(2+) competitively inhibited the activation by Mg(2+), Mn(2+) and Co(2+) for both the tumour and rabbit enzymes. 7. It is concluded that there are no significant differences in metal ion specificity between the tumour and rabbit enzymes. 8. The possible role of metal ions in regulating enzymic and metabolic activities is considered further.

Animals↗
PubMed1983

Regulation of pyruvate kinase expression and growth in mastocytoma cells. I. Initial observations.

The specific activities of pyruvate kinase and phosphofructokinase but not lactate dehydrogenase increase as P-815 mastocytoma cells approach the stationary phase. During this growth period, the rates of uptake of labelled precursors into DNA, RNA and total protein decreases. On the other hand, the pyruvate kinase protein level changes in parallel with activity. Although the K-isozyme is the primary form of pyruvate kinase expressed, some M-type subunit is also present and both forms undergo an increase in specific activity. In addition, pyruvate kinase expression is also elevated by adding cAMP analogues with theophylline, butyrate or conditioned media. This increased level of expression is hypothesized to be a secondary event associated with a differentiation-like-induced expression of the mast cell phenotype.

Animals↗