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PubMed1989

Pyruvate kinase isozymes from the green alga, Selenastrum minutum. I. Purification and physical and immunological characterization.

Pyruvate kinase from the green alga Selenastrum minutum consists of two isoforms (PK1 and PK2) separable by Q-Sepharose chromatography. The two isoforms have been highly purified to respective final specific activities of 42 and 23 (mumol pyruvate produced/min)/mg protein. Purification steps included salt fractionation, anion-exchange, hydrophobic interaction, and gel filtration chromatography. The final enzyme preparations differ significantly in physical and immunological properties. PK1 is heat labile and is completely inactivated following reaction with N-ethylmaleimide. In contrast, PK2 is heat-stable and is only partially inactivated following N-ethylmaleimide treatment. PK1 appears to be homotetrameric with a native molecular mass of about 240 kDa, whereas PK2 appears to be homodecameric with a native molecular mass of approximately 590 kDa. The antigenic reaction of both final PK preparations to rabbit antiserum prepared against homogeneous germinating castor bean endosperm cytosolic pyruvate kinase was tested by immunoprecipitation and Western blotting. The two algal pyruvate kinases are immunologically unrelated as only PK2 cross-reacts with the cytosolic pyruvate kinase antibodies. These data indicate that the S. minutum pyruvate kinase isoforms, PK1 and PK2, are not interconvertible forms of the same protein, but probably represent chloroplastic and cytosolic isozymes, respectively.

Ammonium Sulfate↗
PubMed1995

Cloning of the two pyruvate kinase isoenzyme structural genes from Escherichia coli: the relative roles of these enzymes in pyruvate biosynthesis.

We report the cloning of the pykA and pykF genes from Escherichia coli, which code for the two pyruvate kinase isoenzymes (ATP:pyruvate 2-O-phosphotransferases; EC 2.7.1.40) in this microorganism. These genes were insertionally inactivated with antibiotic resistance markers and utilized to interrupt one or both pyk genes in the E. coli chromosome. With these constructions, we were able to study the role of these isoenzymes in pyruvate biosynthesis.

Cloning, Molecular↗
PubMed1982

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits.

A study was performed to determine whether M1 and M2 pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M1 and M2 pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M1 or M2. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M1 and M2 with V8 protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.

Animals↗
PubMed1981

Isozyme differentiation of aldolase and pyruvate kinase in fetal, regenerating, preneoplastic, and malignant rat hepatocytes during culture.

Aldolase and pyruvate kinase isozymes were investigated in cultured hepatocytes from fetal, regenerating, and 2-acetyl-aminofluorene-fed rat liver as well as in some epithelial liver cell lines. Our results show that: (a) cell proliferation and prolonged expression of specific isozymes were found only in cultured hepatocytes from 17-day old fetuses; (b) the fetal type of pyruvate kinase expressed in regenerating and carcinogen-treated liver was temporarily lost only in cultured hepatocytes from regenerating liver; (c) the adult type of aldolase and pyruvate kinase was absent in one epithelial cell line derived from a carcinogen-treated liver and in the hepatoma tissue cell (HTC) line but was found in the Faza clone of the Reuber H35 cell line during the 50 first passages in vitro; and (d) the isozyme pattern of pyruvate kinase was always more strongly shifted than that of aldolase. The observations suggest that: (a) hepatocytes from carcinogen-treated liver exhibit the same lack of ability to proliferate in primary culture as normal adult hepatocytes; (b) adult hepatocytes can produce fetal isozymes without prior cell division; (c) pyruvate kinase is a stronger marker of dedifferentiation (retrodifferentiation) than aldolase; and (d) regulatory processes of isozyme expression are different during ontogenesis, regeneration, and hepatocarcinogenesis.

Animals↗
PubMed1989

Metabolic effects of high glucose concentrations: inhibition of hepatic pyruvate kinase.

We tested the in vitro effects of various glucose concentrations on the activity of hepatic pyruvate kinase, assayed at subsaturating, near physiological concentration (0.20 mmol/l) of the substrate phosphoenolpyruvate, to detect the "active" form of the enzyme. A 10-min incubation of mouse liver slices (n = 18) with increasing glucose concentrations (5, 10 and 20 mmol/l) resulted in a significant (p less than 0.01), progressive pyruvate kinase inhibition of 15, 28 and 41%, respectively. Similar data were obtained by incubating mouse liver homogenates (n = 7) with glucose, although with this material (which was supplemented with the pyruvate kinase activator fructose-1,6-diphosphate) the inhibition at the highest glucose concentration used was lower (24%, p less than 0.02). Addition of 10 nmol/l insulin during slice incubation (n = 8) prevented by 98% and 69% the inhibition exerted by 10 and 20 mmol/l glucose, respectively. Insulin alone was without effect on the enzyme activity. Glucose might inhibit pyruvate kinase by competing with the activator fructose-1,6-diphosphate. Insulin might overcome the glucose effect by activating pyruvate kinase through the known mechanism of enzyme dephosphorylation. Thus, in decompensated diabetes the high level of blood glucose may contribute, together with the counterregulatory hormones, to inhibit hepatic pyruvate kinase and therefore to stimulate gluconeogenesis.

Animals↗
PubMed2000

Exogenous Mg-ATP induces a large inhibition of pyruvate kinase in intact rat hepatocytes.

Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo.

Adenosine Diphosphate↗
PubMed1993

Pyruvate kinase from Lactobacillus bulgaricus: possible regulation by competition between strong and weak effectors.

The pyruvate kinase from Lactobacillus bulgaricus has been purified to homogeneity. The native enzyme is composed of four probably identical subunits of relative molecular mass M(r) 72,000 +/- 4,000. The unique N-terminal amino acid sequence is homologous to those of other pyruvate kinases, especially of type I and II enzymes from Escherichia coli. The saturation of the pyruvate kinase from Lactobacillus bulgaricus is hyperbolic for ADP and cooperative for the other substrate phospho-enol-pyruvate. The enzyme is strongly activated by glucose-6-phosphate, ribose-5-phosphate, and fructose-6-phosphate, which increase the affinity for phospho-enol-pyruvate. These activators seem to stabilize the same state of the enzyme, since their maximum activations are not additive, but their partial activations can be cumulated. Pyruvate kinase is also weakly activated by AMP and inhibited by fructose-1,6-bisphosphate. However, both AMP and fructose-1,6-bisphosphate act as strong inhibitors in the presence of a strong activator, because these weak effectors suppress the activation by glucose-6-phosphate, ribose-5-phosphate, or fructose-6-phosphate. This mutual exclusion of strong and weak effectors, which appears as an original regulatory mechanism, could reflect either the binding of different effectors to different interacting sites or their competition for a unique polyvalent regulatory site in the pyruvate kinase from Lactobacillus bulgaricus.

Adenosine Diphosphate↗
PubMed1990

The role of inhibition of pyruvate kinase in the stimulation of gluconeogenesis by glucagon: a reevaluation.

We have reexamined the concept that glucagon controls gluconeogenesis from lactate-pyruvate in isolated rat hepatocytes almost entirely by inhibition of flux through pyruvate kinase, thereby making gluconeogenesis more efficient. 1. We tested and refined the 14C-tracer technique that has previously yielded the opposite conclusion, that is, that inhibition of pyruvate kinase is a relatively unimportant mechanism. The tracer procedure, as used by us, was found to be insensitive to the size of the pyruvate pool, and experiments using modifications of the technique to obviate a number of other potential errors support the earlier conclusion that control of pyruvate kinase is not the predominant mechanism. 2. Any stimulation of formation of glucose that results from inhibition of pyruvate kinase is the consequence of elevation of the steady-state concentrations of phosphoenolpyruvate and all subsequent intermediates in the gluconeogenic pathway. During ongoing stimulation of glucose synthesis by glucagon in isolated hepatocytes, the concentrations of all measured intermediate compounds between phosphoenolpyruvate and glucose were elevated except triose phosphates and fructose 1,6-bisphosphate. The failure of these compounds to rise above control levels indicates that not all gluconeogenic reactions beyond pyruvate kinase were accelerated thermodynamically as would occur with predominant control at pyruvate kinase. We conclude, therefore, that although glucagon inhibits flux through the pyruvate kinase reaction, this does not account for most of the stimulation of gluconeogenesis. Major control sites are also within the pyruvate-phosphoenolpyruvate segment and the fructose 1,6-bisphosphate cycle.

Adenosine↗
PubMed2003

Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures.

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry 4, 1518-1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64-fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135-fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34-3.5 and 3.3-0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.

Animals↗
PubMed1992

Cell cycle-associated expression of M2-type isozyme of pyruvate kinase in proliferating rat thymocytes.

During a complete cell cycle of rat thymocytes stimulated by concanavalin A and interleukin 2, the activity and mRNA level of pyruvate kinase reached a maximum (8-12-fold increase) 48 h after stimulation coinciding with the S-phase of the cell cycle. Increases of cellular enzyme activity, pyruvate kinase protein, and mRNA levels are correlated up to 48 h of culture. Afterwards pyruvate kinase activity and mRNA levels decrease, whereas the pyruvate kinase protein continues to increase throughout mitosis. This change of specific pyruvate kinase activity points to a posttranslational modification of the enzyme besides its transcriptional regulation. The presence of the M2-type isozyme was determined by the following methods: (a) native cellulose acetate electrophoresis and activity staining, (b) Northern blot hybridization with M1- and M2-specific cDNA probes, and (c) determination of kinetic parameters. The isozyme pattern did not change during the cell cycle progression. The induction of pyruvate kinase is completely abolished by 2-difluoromethylornithine-mediated polyamine depletion. However, the proportion of hybridizable pyruvate kinase mRNA was not affected. These data suggest the requirement of polyamines for efficient translation rather than transcription during cell growth.

Animals↗
PubMed1976

Regulation in vitro of rat liver pyruvate kinase by phosphorylation-dephosphorylation reactions, catalyzed by cyclic-AMP dependent protein kinases and a histone phosphatase.

1. Cyclic-AMP dependent protein kinases, resolved by chromatography on DEAE-cellulose and hydroxylapatite, catalysed the phosphorylation of rat liver pyruvate kinase and calf thymus histones by [gamma32P]ATP. [32P]phosphopeptides, from acid hydrolysates of pyruvate kinase phosphorylated by the different protein kinase fractions, displayed identical electrophoretic patterns. Phosphorylation inhibited pyruvate kinase activity. 2. Full activity was restored when phosphorylated pyruvate kinase was dephosphorylated by a histone phosphatase from the soluble fraction of rat liver. These results are consistent with the hypothesis that pyruvate kinase is regulated by phosphorylation-dephosphorylation reactions.

Animals↗
PubMed1999

A novel factor binding to the glucose response elements of liver pyruvate kinase and fatty acid synthase genes.

Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z. , Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268, 12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.

Animals↗
PubMed1982

Regulation by glucagon of hepatic pyruvate kinase, 6-phosphofructo 1-kinase, and fructose-1,6-bisphosphatase.

Glucagon stimulates gluconeogenesis in part by decreasing the rate of phosphoenolpyruvate disposal by pyruvate kinase. Glucagon, via cyclic AMP (cAMP) and the cAMP-dependent protein kinase, enhances phosphorylation of pyruvate kinase, phosphofructokinase, and fructose-1,6-bisphosphatase. Phosphorylation of pyruvate kinase results in enzyme inhibition and decreased recycling of phosphoenolpyruvate to pyruvate and enhanced glucose synthesis. Although phosphorylation of 6-phosphofructo 1-kinase and fructose-1,6-bisphosphatase is catalyzed in vitro by the cAMP-dependent protein kinase, the role of phosphorylation in regulating the activity of and flux through these enzymes in intact cells is uncertain. Glucagon regulation of these two enzyme activities is brought about primarily by changes in the level of a novel sugar diphosphate, fructose 2,6-bisphosphate. This compound is an activator of phosphofructokinase and an inhibitor of fructose-1,6-bisphosphatase; it also potentiates the effect of AMP on both enzymes. Glucagon addition to isolated liver systems results in a greater than 90% decrease in the level of this compound. This effect explains in large part the effect of glucagon to enhance flux through fructose-1,6-bisphosphatase and to suppress flux through phosphofructokinase. The discovery of fructose 2,6-bisphosphate has greatly furthered our understanding of regulation at the fructose 6-phosphate/fructose 1,6-bisphosphate substrate cycle.

Animals↗
PubMed1980

Biosynthesis of rat liver pyruvate kinase. Measurement of enzyme lifetime and the rate of synthesis at weaning.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of immunoprecipitates of liver cytosol with anti-(L-type pyruvate kinase) serum revealed proteins of mol.wt. 56 000 and 42 000 in addition to the heavy and light chains. The ratio of the 56 000 mol.wt. to the 42 000 mol.wt. protein increased under dietary conditions that resulted in an increase in the apparent specific activity of hepatic pyruvate kinase. The 42 000 mol.wt. protein was removed from immunoprecipitates if the liver cytosol was partially purified by pH precipitation and (NH4)2SO4 fractionation before addition of the antiserum. This technique may be used to analyse the formation of pure L-type pyruvate kinase in liver. By using H14CO3-labelling, the t1/2 of L-type pyruvate kinase was estimated as 75 +/- 1.7 h in post-weaned high-carbohydrate-diet-fed rats. Before weaning there was little immunoreactive pyruvate kinase in rat liver cytosol. Induction began between 6 and 24 h after weaning and reached a maximum value 120 h after weaning. When clearly enhanced total pyruvate kinase activity was first observed at 24 h post-weaning, the apparent specific activity of hepatic pyruvate kinase was considerably lower than the specific activity of the pure isolated enzyme. When the induction of L-type pyruvate kinase was monitored by the incorporation of L-[4,5-3H]leucine, the maximum rate of synthesis occurred 24--48 h after weaning. After this period synthesis declined, indicating a relatively slow turnover of the enzyme once the enzyme concentration was established in the liver.

Animals↗
PubMed1980

Pyruvate kinase deficiency hemolytic anemia: enzymatic characterization studies in twelve patients.

Erythrocyte pyruvate kinase from twelve patients with hereditary erythrocyte pyruvate kinase (PK) deficiency was investigated according to the recommended methods for the characterization of red cell pyruvate kinase variants. Family members were also studied. Abnormalities were frequently noted in the affinity for the substrate, phosphoenolpyruvate; allosteric activator, fructose-1,6-diphosphate; allosteric inhibitor, ATP; and also in heat stability and electrophoretic pattern. Several different PK variants were identified. Polyacrylamide gel electrophoresis revealed the presence of immature activity bands in the red cells of some patients. These bands presumably represent residuals of isozymes produced during the evolution of erythrocyte PK, and may occur as a compensatory mechanism for the defective isozyme.

Adenosine Triphosphate↗
PubMed1981

An allele (Pk-1b) from wild-caught mice that affects the activity and kinetics of erythrocyte and liver pyruvate kinase.

A true breeding strain was made from a wild-caught mouse with low erythrocyte pyruvate kinase (E.C. 2.7.1.40) activity. This variation showed additive inheritance and segregated as an allele at a single locus (Pk-1b). Mice homozygous for the reduced blood pyruvate kinase activity cosegregated for reduced liver activity. In both these tissues the variant enzyme had a lowered heat stability and reduced Km values for ADP. An increased stimulation by FDP was also detected in the liver pyruvate kinase. No difference in the isoelectric point of the variant enzyme in either erythrocyte or liver was observed when compared with the enzyme from C57BL mice (Pk-1a/Pk-1a). It is concluded that Pk-1 is the structural gene for the erythrocyte and the major liver pyruvate kinase. No other tissue pyruvate kinase showed altered characteristics.

Alleles↗
PubMed1969

Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties.

1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the K(m) for phosphoenolpyruvate. 2. A ;cold' variant of the enzyme has hyperbolic kinetics and exhibits a minimal K(m) for substrate at 5 degrees . At physiological concentrations of phosphoenolpyruvate the ;cold' enzyme is active only below 10 degrees . A ;warm' pyruvate kinase has a minimal K(m) for substrate at about 12 degrees . This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9 degrees . 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4-12 degrees . 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the ;warm' pyruvate kinase and the ;cold' pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the ;warm' enzyme is converted into the ;cold' enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.

Animals↗
PubMed1986

Transcriptional and post-transcriptional regulation of L-type pyruvate kinase gene expression in rat liver.

The effects of starvation, refeeding a diet high in carbohydrate, administration of glucagon and cyclic AMP, thyroidectomy, and adrenalectomy on transcription of the gene for liver L-type pyruvate kinase and on the accumulation of cytoplasmic mRNA for L-type pyruvate kinase were investigated in rat. Transcription of the gene was undetectable in either fasted or protein-fed rats. Refeeding fasted rats a carbohydrate-rich diet stimulated an increase in L-type pyruvate kinase mRNA, preceded by an increase in the gene transcription. Transcription was maximal at 12 h of refeeding, decreasing to 10% of maximum at 72 h. The level of L-type pyruvate kinase mRNA remained constant at 50% of maximum for at least 120 h. Neither thyroidectomy nor adrenalectomy affected gene transcription in fasted rats refed the carbohydrate-rich diet, despite a decrease in mRNA abundance to 40 and 20%, respectively, of controls fed a normal diet. Glucagon or cyclic AMP totally blocked the increase in transcription of the L-type pyruvate kinase gene caused by feeding a carbohydrate-rich diet to previously fasted rats. Nevertheless, the level of L-type pyruvate kinase mRNA remained high for 3 h after glucagon administration. After 3 h, the mRNA decreased rapidly with a half-life less than 1 h. Thus, expression of the gene for L-type pyruvate kinase is regulated at both transcriptional and post-transcriptional levels. The transcription is regulated by two major effectors, one positive, namely carbohydrates, and one negative, namely glucagon (via cyclic AMP). Both agents probably act at the level of the mRNA stability as well. Glucocorticoids and thyroid hormones do not regulate transcription of the gene for L-type pyruvate kinase but do appear to be required for a normal accumulation of the transcripts in the cytoplasm.

Adrenalectomy↗