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PubMed1984

Studies on the regulation of hepatic pyruvate kinase synthesis in neonatal rats.

The L- and M2-type pyruvate kinase from the liver of 1-day old rats demonstrated no significant activation nor inhibition by treatment with cyclic AMP, glucagon or insulin. Neither was there any change in their isozymic composition. By means of incorporation with [3H]leucine followed by immunoprecipitation, the rates of synthesis of both the L- and M2-type pyruvate kinase were not considerably affected by all three modulators. Insulin and glucagon do not direct an immediate change in the synthesis of liver pyruvate kinase and a fluctuation in the insulin/glucagon ratio is not a probable signal for regulating the isozymic expression in the neonatal period.

Animals↗
PubMed2005

The effects of the interactions between amino acids on pyruvate kinase activity from the brain cortex of young rats.

Considering that pyruvate kinase activity, a crucial enzyme for glucose metabolism and energy liberation in brain, may be regulated by some amino acids, it is possible that diminution of this enzyme activity may contribute to the brain damage caused by amino acids accumulated in metabolic diseases, such as phenylalanine, tryptophan and cystine. Therefore, the present study was undertaken to investigate the effect of these amino acids on pyruvate kinase activity in the brain cortex of rats. We also investigated the effect of serine and alanine on pyruvate kinase activity in the same tissue. The results suggested that phenylalanine, tryptophan, cystine, alanine, and serine act at the same site on the enzyme, phenylalanine, tryptophan, and cystine causing inhibition, and alanine and serine preventing this effect. Cystine also inhibited the enzyme activity through a different mechanism, possibly acting on the enzyme thiol groups. Considering that this enzyme is a target for amino acids accumulated in some metabolic diseases of amino acid metabolism, it is possible that its inhibition may contribute to the brain damage found in these diseases.

Amino Acid Metabolism, Inborn Errors↗
PubMed1974

The preparation and properties of pyruvate kinase from yeast.

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn(2+) ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn(2+)-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).

Benzoates↗
PubMed1981

Regulation of pyruvate kinase by 6-phosphogluconate in isolated hepatocytes.

Isolated liver parenchymal cells from rats fed a 65% sucrose diet for 14 days were incubated in the presence and absence of 10(-6) M glucagon. The pyruvate kinase obtained from homogenates of the glucagon-treated cells displayed and increased Ks 0.5 for phosphoenolpyruvate (P-enolpyruvate), as well as an increased Ka 0.5 for 6-phosphogluconate (6-P-gluconate), compared to pyruvate kinase from untreated cells. Additionally, glucagon treatment decreased the maximal stimulation of pyruvate kinase by 6-P-gluconate by approximately two-thirds and decreased the Hill coefficient value of pyruvate kinase for 6-P-gluconate from 1.76 to 1.56. 6-Aminonicotinamide, an inhibitor of 6-P-gluconate dehydrogenase, increased 6-P-gluconate levels in isolated liver parenchymal cells three- to sevenfold, depending on the substrates present. The flux of P-enolpyruvate through pyruvate kinase was increased from 18 to 40% in these preparations and was highly correlated with the increase in 6-P-gluconate levels. The results suggest that 6-P-gluconate could regulate pyruvate kinase activity in the intact liver parenchymal cell. Furthermore, the activator would be of greatest importance in the lipogenic animal.

Animals↗
PubMed2005

Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis.

Four isozymes of pyruvate kinase are differentially expressed in human tissue. Human pyruvate kinase isozyme M2 (hPKM2) is expressed in early fetal tissues and is progressively replaced by the other three isozymes, M1, R, and L, immediately after birth. In most cancer cells, hPKM2 is once again expressed to promote tumor cell proliferation. Because of its almost ubiquitous presence in cancer cells, hPKM2 has been designated as tumor specific PK-M2, and its presence in human plasma is currently being used as a molecular marker for the diagnosis of various cancers. The X-ray structure of human hPKM2 complexed with Mg(2+), K(+), the inhibitor oxalate, and the allosteric activator fructose 1,6-bisphosphate (FBP) has been determined to a resolution of 2.82 A. The active site of hPKM2 is in a partially closed conformation most likely resulting from a ligand-induced domain closure promoted by the binding of FBP. In all four subunits of the enzyme tetramer, a conserved water molecule is observed on the 2-si face of the prospective enolate and supports the hypothesis that a proton-relay system is acting as the proton donor of the reaction (1). Significant structural differences among the human M2, rabbit muscle M1, and the human R isozymes are observed, especially in the orientation of the FBP-activating loop, which is in a closed conformation when FBP is bound. The structural differences observed between the PK isozymes could potentially be exploited as unique structural templates for the design of allosteric drugs against the disease states associated with the various PK isozymes, especially cancer and nonspherocytic hemolytic anemia.

Allosteric Regulation↗
PubMed1973

Phenylketonuria: phenylalanine inhibits brain pyruvate kinase in vivo.

The hypothesis that brain damage in phenylketonuria is related to inhibition of pyruvate kinase by phenylalanine was examined in rat brain in vivo. One hour after a single injection of phenylalanine into the rat, the brains were removed and completely frozen in less than a second. The concentration of phenylalanine in the brain was comparable to that found in phenylketonuric patients. Changes in brain glycolytic intermediates were consistent with inhibition of pyruvate kinase in vivo. The inhibition of pyruvate kinase was apparently compensated for by an increase in phosphoenolpyruvate; no decrease in adenosine triphosphate or creatine phosphate was found.

Adenosine Diphosphate↗
PubMed1980

Dietary alteration of translatable mRNA sequences coding for rat liver pyruvate kinase.

Poly(A+) RNA (RNA containing a polyadenylic acid sequence) was isolated from individual livers of rats fed standard lab chow, fasted, or fasted and refed a high carbohydrate diet. The level of functional mRNA coding for pyruvate kinase was assayed using a rabbit reticulocyte in vitro translation system. The total 35S incorporation into newly synthesized liver pyruvate kinase was measured and compared to 35S incorporation into albumin, total trichloroacetic acid-precipitated proteins, and released polypeptide chains. The relative level of mRNA coding for liver pyruvate kinase decreases almost 60% upon fasting and increases approximately 15-fold upon refeeding with a high carbohydrate diet for 24 h. These observed changes in the amount of mRNA coding for liver pyruvate kinase agree with the previously reported changes in the relative rates of liver pyruvate kinase synthesis measured in vivo during these dietary stresses. Thus, it is suggested that the alterations in the amount of pyruvate kinase in liver in response to these dietary stresses primarily result from alteration in the amount of functional mRNA coding for the enzyme.

Animals↗
PubMed1995

[Prenatal diagnosis of homozygous pyruvate kinase deficiency].

Two consecutive cases of severe neonatal anaemia due to severe deficiency in pyruvate kinase were observed in the same sibhood. The first child died one hour after birth and the second required major transfusion support. Pyruvate kinase deficiency is a rare cause of congenital anaemia with recessive autosomic inheritance. Clinically, this deficiency has a very variable expression, and neonatal forms are not always very severe. Several variant molecules in pyruvate kinase deficiency have been described. Recent progress in our understanding of the gene would suggest the possibility of new diagnostic and prognostic approaches.

Anemia, Hemolytic, Congenital↗
PubMedDate not supplied

[Study of some properties of rat myocardial pyruvate kinase in adrenaline myocarditis].

Adrenaline myocarditis in rats was accompanied by a decrease in relative activity of pyruvate kinase from hyaloplasm of heart muscle, especially pronounced within 6-13 hrs after adrenaline administration. This phenomenon was probably due to an increase in content of protein in hyaloplasm, but not to an alteration in the enzyme activity. This conclusion was confirmed by studies of some properties of pyruvate kinase. Under these pathological conditions the Km of phosphoenol pyruvate was of the same order of magnitude as in normal state; 1-phenylalanine was characterized as an inhibitor of non-competitive type tomards phosphoenol pyruvate, and palmitate did not exhibit the inhibitory effect on pyruvate kinase as it was found in normal state. The effectiveness of the enzyme inhibition by L-phenylalanine, protection of the enzyme against this amino acid effect by L-alpha-alanine, L-serine and L-cysteine, and also the degree of activation of pyruvate kinase by the amino acids under adrenaline myocarditis were similar to those phenomena observed in normal state.

Amino Acids↗
PubMed1987

Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis.

1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of microM concentrations of fructose-1,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.

Animals↗
PubMed1988

Production of a specific antibody against pyruvate kinase type M2 using a synthetic peptide.

The pyruvate kinase isozymes M1 and M2 are structurally and immunologically closely related. To obtain an antibody which discriminates between these two forms, a synthetic tetradecapeptide with a sequence specific for pyruvate kinase type M2 from rats was constructed. Antisera from rabbits, immunized with this peptide, reacted specifically with the M2-type holoenzyme of both rat and human origin, and did not cross-react with the M1-type isozyme. This was established by immunoblot analysis, both under dissociating and non-dissociating conditions.

Amino Acid Sequence↗
PubMed1981

Glucose 6-phosphate activation of pyruvate kinase from Mycobacterium smegmatis.

1. Activation of glucose 6-phosphate is one of the unique properties of pyruvate kinase from Mycobacterium smegmatis. 2. Pyruvate kinase, partially purified from ultrasonic extracts of the mycobacteria by (NH4)2SO4 fractionation, exhibited sigmoidal kinetics at various concentrations of phosphoenolpyruvate, with a high degree of co-operativity (Hill coefficient, h = 3.7) and S0.5 value of 1.0 mM. 3. In the presence of glucose 6-phosphate, the degree of co-operativity shown by the phosphoenolpyruvate saturation curve was decreased to h = 2.33 and the S0.5 value was lowered to 0.47 mM. 4. The enzyme was activated by AMP and ribose 5-phosphate also, but the activation constant was lowest with glucose 6-phosphate (0.24 mM). 5. The enzyme was strongly inhibited by ATP at all phosphoenolpyruvate concentrations. The concentrations of ATP required to produce half-maximal inhibition of enzyme activity at non-saturating (0.2 mM) and saturating (2 mM) phosphoenolpyruvate concentrations were 1.1 mM and 3 mM respectively. 6. The inhibition of ATP was partially relieved by glucose 6-phosphate. 7. The enzyme exhibited Michaelis-Menten kinetics with ADP as the variable substrate, with an apparent Km of 0.66 mM. 8. The enzyme required Mg2+ or Mn2+ ions for activity. It was not activated by univalent cations. 9. The kinetic data indicate that under physiological conditions glucose 6-phosphate probably plays a significant role in the regulation of pyruvate kinase activity.

Enzyme Activation↗
PubMed1975

A new variant of erythrocyte pyruvate kinase - PK "maebashi".

A new case of erythrocyte pyruvate kinase deficiency was described. - A 9 year-old male patient was hospitalized because of anaemia, jaundice and splenomegaly. Diagnosis was made primarily on the basis of the erythrocyte enzyme studies. Because the pyruvate kinase of this patient demonstrated certain different characteristics from the other variants described previously, it was tentatively designated PK "Maebashi".

Adenine Nucleotides↗
PubMed1986

Regulation of pyruvate kinase in cultured rat hepatocytes. Influence of glucose, ethanol, glucagon, and dexamethasone.

The hormonal regulation and molecular forms of Type L pyruvate kinase were investigated in rat hepatocytes maintained in primary culture. Five isoelectric forms of the enzyme subunit were identified by isoelectric focusing in 8 M urea. Immediately after pulse labeling rat hepatocytes with [35S]methionine radioactivity was observed in one major (D-band) and one minor (I-band) peptide band. These isoelectric forms were shown to be dephosphorylated forms of the subunit. Acute administration of 0.1 microM glucagon was accompanied by disappearance of the D- and I-bands and appearance of two additional forms (P- and A-bands, respectively). These latter two forms were demonstrated to be phosphorylated forms of the subunit. A fifth isoelectric form of the pyruvate kinase subunit (B-band) was identified by immunolocation; however, incorporation of radioisotope into this band was low. Chronic administration of glucagon or dexamethasone had no significant influence on the molecular properties of pyruvate kinase. However, novel observations concerning the influence of glucose and ethanol on the phosphorylation state of the enzyme were made. When hepatocytes were maintained at 5.5 mM glucose for 24-48 h, the activity ratio for pyruvate kinase decreased from 0.65 to 0.40 and the enzyme became partially phosphorylated. Raising the glucose concentration to 28 mM prevented or rapidly reversed the phosphorylation state of the enzyme. Administration of low concentrations of ethanol (1-20 mM) caused a decline in the activity ratio of pyruvate kinase in the presence of both 5.5 and 28 mM glucose. These latter observations concerning the influence of glucose and ethanol are the first demonstrating that nutrients or metabolites alter the phosphorylation state of the enzyme in the absence of hormonal stimuli.

Animals↗
PubMed1986

Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence.

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozyme of pyruvate kinase for the first time. The deduced amino acid sequence of the L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with M1-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M1-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M1 type except for one amino acid substitution.

Amino Acid Sequence↗
PubMed1999

Pyruvate kinase as a microtubule destabilizing factor in vitro.

Endogenous control of microtubule dynamism is essential in many cell types. Numerous microtubule-adhering proteins stabilize the polymer status, while very few protein factors are described with opposite effects. The brain- and muscle-specific M1 isoform of the enzyme pyruvate kinase is investigated here in this respect. Three pieces of evidence indicate antimicrotubular effects of this protein. (1) Pyruvate kinase inhibits taxol-induced tubulin polymerization into microtubules as revealed by turbidimetry. (2) Pelleting experiments show that pyruvate kinase partially disassembles taxol-stabilized microtubules into less sedimentable oligomers leading to the appearance of tubulin in the supernatant fractions. (3) Electron microscopy reveals the kinase-induced formation of great amounts of thread-like tubulin oligomers which tend to accumulate in a light/less sedimentable fraction. Immunoelectron micrographs using labeled antibody against pyruvate kinase provide evidence for the binding of pyruvate kinase to the thread-like oligomeric forms. The present data allow the assumption that pyruvate kinase may display multiple regulatory functions as a glycolytic control enzyme and as a modulator of microtubule dynamism.

Animals↗
PubMed1977

Differentiation in vivo and in vitro of pyruvate kinase isozymes in rat muscle.

The M type isozymes of Pyruvate-kinase have been studied by isoelectrofocusing in thin layer acrylamide ampholine gel, during the ontogeny of rat muscle in vivo and during the differentiation in vitro of myoblasts of a line established by Yaffe. In both cases, multiple subbands have been seen; the most acid (pHi 5.2) was the predominant band in myoblasts and in fetal muscle at the 15th day. Several more cathodic bands appear sequentially in vitro as in vivo, one of them corresponding to the "M2" or "K" band (predominant in kidney). The most cathodic band M1 (pHi 7.3), characteristic of the adult muscle, appears at the 9th day of culture in vitro in multinucleated myotubes and at the 20th day of fetal life in vivo. Kinetic results confirm these electrofocusing results, showing in fetal muscle and in myoblasts a sigmoid saturation curve of pyruvate kinase activity with phosphoenolpyruvate as substrate. This allosteric kinetic is progressively replaced by a Michaëlian kinetics in vitro as in vivo. Consequently, the studies in vitro may serve as a model for myogenesis in vivo, and may contribute to the understanding of the significance of the multiple forms of pyruvate-kinase during this myogenesis.

Aging↗