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PubMed1987

Hormonal regulation of L-type pyruvate kinase in rat liver cells in culture.

An immortalized rat liver cell line (RLC) expresses two isozymes of pyruvate kinase, the adult liver or L-type isozyme and an M-type isozyme presumed to be the M2-type. In RLC cells incubated in serum-free medium, the addition of 0.1 microM insulin maintained the initial level of L-type pyruvate kinase when it was high and induced the L-type isozyme when it was low. The addition of 1.0 mM dibutyryl cAMP and 0.5 mM theophylline decreased the L-type isozyme, even in the presence of insulin. The amount of M2-type isozyme was relatively constant under the conditions used. Regulation of the amount of L-type pyruvate kinase by both insulin and cAMP occurred primarily through changes in the rate of L-pyruvate kinase protein synthesis and translatable mRNA levels. These results are consistent with the in vivo observations that both insulin and glucagon regulate the rate of L-pyruvate kinase gene transcription and that cAMP is the dominant regulator of L-pyruvate kinase gene expression.

Animals↗
PubMed1987

Liver specific pyruvate kinase in pheasants Phasianus colchicus.

While in chickens, the existence of a liver form of pyruvate kinase is controversial, the liver form of pyruvate kinase in pheasants, murres and puffins is electrophoretically distinct from that in muscle, brain, kidney, lung and small intestine. Although the forms in lungs, muscle, heart, brain and small intestine could not be reliably separated by electrophoresis, the functional characteristics of the lung and muscle forms of pyruvate kinase in the pheasant are distinct and can be classified as K and M isozymes respectively. Our data suggest that these birds possess at least three distinct isozymes of pyruvate kinase.

Animals↗
PubMed1982

Affinity labeling of rabbit muscle pyruvate kinase with dialdehyde-ADP.

Periodate-oxidized ADP (dialdehyde-ADP) inactivates rabbit muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) and combines irreversibly to the enzyme. This inactivation is first-order with respect to dialdehyde-ADP and follows saturation kinetics, indicating that the enzyme first forms a reversible complex with the inactivator. Low Mg2+ concentrations stimulate the rate of inactivation, while higher concentrations have a protective effect. ADP and ATP, especially in the presence of Mg2+, protect very strongly against inactivation, while phosphoenolpyruvate and pyruvate are less effective. Dialdehyde-ADP is not a substrate, but acts as competitive inhibitor of ADP, with a KI of 4.5 mM. The analog has somewhat lower affinity to the enzyme than Mg-ADP, which has a Kd of 1.2 mM. Based on kinetic data, it is shown that one molecule of reagent must combine per enzyme active site in order to inactivate the enzyme. Incorporation of [14-C]dialdehyde-ADP to the enzyme and treatment of the data by the Tsou plot shows that 6-7 residues per subunit react with the modifier, two of them being essential for activity. From the evidence presented it is concluded: (1) dialdehyde-ADP behaves as an affinity label of rabbit muscle pyruvate kinase; (2) the inactivator binds probably to lysine residues at or near the active site, forming morpholine-like structures, and (3) the enzyme possesses two modifiable groups essential for activity, the reaction of one of them being sufficient to cause total loss in activity.

Adenosine Diphosphate↗
PubMed1979

Two forms of pyruvate kinase in Escherichia coli. A comparison of chemical and molecular properties.

The two forms of pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Escherichia coli have been purified from the same cultures and crystallized. A modified procedure for the purification of type I pyruvate kinase is described. Molecular weight, subunit structure, amino acid composition, NH2-terminal amino acid, maps of tryptic peptides and conditions for crystallization have been determined for the two forms. A comparison of these data shows that the two forms are different proteins, each being a tetramer of identical subunits.

Amino Acids↗
PubMed1983

Structural and kinetic differences between the M2 type pyruvate kinases from lung and various tumors.

The kinetic and structural properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that from Rous sarcoma virus-transformed chicken fibroblasts, have been compared. The "tumor enzyme" is characterized by a low affinity for phosphoenolpyruvate, pronounced serine activation, and strong alanine inhibition as compared to the "lung type". In contrast to the rat lung enzyme, which is not affected by serine, the chicken lung enzyme is slightly activated by serine. The serine metabolites phosphoserine and glycine do not activate the "tumor type M2 pyruvate kinase", but L-alpha-glycerophosphorylcholine and phosphatidylserine do slightly activate at physiological concentrations. Studies with substances structurally related to serine reveal that the hydroxyl group of serine is a prerequisite for the activation and that the amino and carboxyl groups determine the affinity of the "tumor type M2 pyruvate kinase" for serine. Two different fragmentation methods (CNBr-cleavage and V-8 proteolysis) and two different methods for separation of the resulting peptide fragments (polyacrylamide gel isoelectric focussing and SDS-polyacrylamide electrophoresis) indicated a high degree of homology between the type M2 pyruvate kinases from lung and tumors as well the type M1 from muscle. Each type of pyruvate kinase, however, contains one or two unique protein fragments which are characteristic for its type. We have termed those fragments L (lung), M (muscle), and T (tumor).

Animals↗
PubMed1984

The influence of inorganic phosphate and ATP on the kinetics of bovine heart muscle pyruvate kinase.

The mechanism of activation by inorganic phosphate and ATP of cardiac muscle pyruvate kinase was studied with the aid of steady-state kinetics. The enzyme was purified to homogeneity to a final specific activity of 400 units/mg (phosphate buffer, pH 7.6, 25 degrees C). At pH 7.6 the enzyme displays Michaelis-Menten kinetics with respect to both its substrates, phosphoenolpyruvate and ADP. Substrate kinetic constants are: app.Km(phosphoenolpyruvate) = 0.04 mM, app.Km(ADP) = 0.22 mM. Under the conditions used in the standard assay the specific activity is greatly enhanced by inorganic phosphate (50 mM) or ATP (2.5 mM). Each of these modifiers, acting separately, increases the Vmax without seriously affecting Michaelis constants and Hill coefficients. In the presence of both Pi and ATP, only a decrease in Vmax was observed. The kinetics of activation by inorganic phosphate of pyruvate kinase was examined. Studying the effect of varying concentrations of Pi on the initial rate we obtained a hyperbolic saturation curve with the app.Km(Pi) = 20 mM and Vmax = 167 units/mg. The evidence is presented that inorganic phosphate is a substrate for a side reaction catalyzed by cardiac pyruvate kinase. It is shown that in the presence of pyruvate, inorganic phosphate and ATP in the assay system, Pi is incorporated into acid-labile products of this reaction, inorganic pyrophosphate being one of them. These findings indicate the existence of an alternative reaction catalyzed by pyruvate kinase by which energy may be stored in the form of inorganic pyrophosphate.

Adenosine Triphosphate↗
PubMed1992

Phosphorylation of pyruvate kinase type K is restricted to the dimeric form.

In the absence of glycolytic intermediate, fructose-1,6-bisphosphate, pyruvate kinase type K exists in the dimeric form and is readily phosphorylated, whereas in the same sample and the same conditions pyruvate kinase type M is present as a tetramer and is not phosphorylated. Addition of fructose-1,6-bisphosphate results in the association of dimeric K2 molecules to a tetrameric K4 enzyme as determined by gel filtration and cellulose acetate electrophoresis, with concomitant loss of the capacity of the K isozyme to become phosphorylated. Phosphorylated K2 dimers can also tetramerize, but with a low recovery of the radiolabel, suggesting a fructose-1,6-bisphosphate induced dephosphorylation or selective degradation. The dimeric K isozyme is enzymatically active; inactive K-type monomers can be detected by immunoblot analysis in the absence of fructose-1,6-bisphosphate, but no phosphorylated pyruvate kinase is present in this fraction. The formation of K4 tetramers can not be accomplished by the substrate phosphoenolpyruvate. Fructose-1,6-bisphosphate is an allosteric activator of pyruvate kinase type K and induces hyperbolic saturation curves for phosphoenolpyruvate. In contrast, in the absence of effectors, pyruvate kinase type M exhibits Michaelis-Menten kinetics, but sigmoidal curves can be induced by the amino acid phenylalanine. However, even in the presence of phenylalanine, the M-type maintained its tetrameric configuration and did not serve as a substrate in the phosphorylation reaction. These findings argue for the importance of subunit interaction in the regulation of phosphorylation of pyruvate kinase.

Astrocytoma↗
PubMedDate not supplied

Prevalence of erythrocyte pyruvate kinase deficiency and normal values of enzyme in a Turkish population.

A pyruvate kinase deficiency prevalence study and determination of the normal levels of the enzyme were performed in Antalya city, Turkey. Heparinized blood samples obtained from a representative population of the Antalya province (617 women and 573 men) were tested for pyruvate kinase deficiency by qualitative and quantitative tests between April 1992 and March 1994. The mean pyruvate kinase activity was found to be 19.8 +/- 4.0 IU/g Hb whereas the enzyme activity of deficient cases varied between 7.5 and 12.2 IU/g Hb. Taking into account that pyruvate kinase deficiency is the second most common cause of nonspherocytic congenital hemolytic anemia, detection of deficient cases by genetic screening tests appears to be an informative clinical indicator of hemolytic anemia.

Erythrocytes↗
PubMed1983

The isolation, characterization, and sequence of the pyruvate kinase gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae gene encoding the glycolytic enzyme pyruvate kinase has been isolated by complementation of a pyk mutant with DNA from a wild type yeast genomic library. Pyruvate kinase enzyme activity is 20-fold higher in the transformant compared to the parental strain and is glucose inducible. The cloned gene has been localized by hybridization of DNA fragments to yeast poly(A+) RNA and by complementation of the mutant defect with select subclones. A DNA sequence of 2885 nucleotides encoding a protein of 499 amino acids is reported. A polypeptide chain of 34 residues of the deduced yeast amino acid sequence closely resembles a peptide sequence at the ADP binding site of bovine muscle pyruvate kinase. The 5' end of the pyruvate kinase mRNA has been mapped and starts within the DNA sequence CAAG at -38 to -27 nucleotides upstream from the first ATG. We note that the sequence PyAAPu in this region appears to be a common consensus site for yeast RNA polymerase II transcriptional starts.

Amino Acids↗
PubMed1997

[Erythrocyte pyruvate kinase--an enzyme that may have an influence on oxygen transport to tissues].

Hemoglobin, the critical protein in the delivery of oxygen to mammalian tissues, is poorly adapted to that function. This awkward situation is remedied by the presence in the red cell of 5 to 7 mM 2.3 DPG, which binds to Hb competitively with oxygen and reduces oxygen affinity. How the levels of 2.3 DPG in the red cell are regulated is an important question that has not yet been fully answered. The best established correlation with 2.3 DPG concentration in red cells is the activity of the enzyme pyruvate kinase. Inverse relationship between 2.3 DPG content and pyruvate kinase activity is the result of two conditions within the erythrocyte. The metabolites between FBP and PEP are in a state of quasi equilibrium because the activity of pyruvate kinase is so much lower than the activities of other enzymes in the pathway. Furthermore, pyruvate kinase operates, in vivo at a PEP concentration well below the Km concentration. In consequence, an increase in the glycolytic rate or inhibition of pyruvate kinase causes an increase in PEP concentration. Increases in PEP levels lead to increases in the levels of 2.3 DPG, and hence to increase in the level of 2.3 DPG via the 2.3 DPG synthase reaction. This relationship is demonstrated by the frequent occurrence of elevated levels of 2.3 DPG in pyruvate kinase deficient erythrocytes and by the decreased levels of 2.3 DPG and PEP which are observed in erythrocytes containing a pyruvate kinase with abnormally high activity at low PEP levels. It is thus clear that control of pyruvate kinase activity is a means to the control of oxygen delivery by the erythrocyte. It remains to be discovered whether any of the observed variations in human PK activity are due to reversible posttranslational modification and whether the potential for control of oxygen delivery via changes in pyruvate kinase activity is made use of the normal human adult. The availability of human full length of cDNA for PK should accelerate our understanding of he structure-function relationships of PK deficiency and enhance the possibility of gene therapy for seriously affected PK patients.

Adult↗
PubMed1983

Diminution of stationary enzyme activities at increases of pyruvate kinase concentration in a reconstituted enzyme system.

In a homogeneous and open enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose 6-phosphate isomerase the consequences of variations of the enzyme concentrations on the stationary enzyme activities have been investigated. An unexpected behavior was observed upon variation of the maximum activity of pyruvate kinase. Depending on the experimental conditions an increase of the concentration of pyruvate kinase resulted either in a diminution or in a stimulation of the stationary activity of this enzyme. An increase of the maximum activity of phosphofructokinase, however, stimulates both the activities of phosphofructokinase and pyruvate kinase. The experimental results are interpreted in terms of a mathematical model, based on the kinetic properties of the enzymes involved. The correlation between the observed changes of the activities of phosphofructokinase and pyruvate kinase and the appearance of multiple stationary states is discussed.

Adenylate Kinase↗
PubMed1974

Kinetic properties of rat liver pyruvate kinase at cellular concentrations of enzyme, substrates and modifiers.

Kinetic properties of rat liver pyruvate kinase type I at pH7.5 and 6.5 were studied with physiological ranges of substrates, modifiers and Mg(2+) concentrations at increasing enzyme concentrations, including the estimated cellular concentrations (approx. 0.1mg/ml). Enzyme properties appear unaffected by increased enzyme concentration if phosphoenolpyruvate, fructose 1,6-diphosphate and inhibitors are incubated with enzyme before starting the reaction with ADP. Our data suggest that minimum cellular concentrations of MgATP and l-alanine provide virtually complete inhibition of pyruvate kinase I at pH7.5. The most likely cellular control of existing pyruvate kinase I results from the strong restoration of enzyme activity by the small physiological amounts of fructose 1,6-diphosphate. Decreasing the pH to 6.5 also restores pyruvate kinase activity, but to only about one-third of its activity in the presence of fructose 1,6-diphosphate. Neither pyruvate nor 2-phosphoglycerate at cellular concentrations inhibit the enzyme significantly.

Adenosine Diphosphate↗
PubMed1989

Expression of pyruvate kinase in astrocytes induced to differentiate in vitro.

Astrocytes maintained in a chemically defined media undergo differentiation and a parallel increase in pyruvate kinase specific activity. These changes are accompanied by a shift in the isoelectrofocusing pattern, but not by expression of pyruvate kinase M4, the characteristic adult rat brain isozyme. Thus, this chemically defined media lacks a substance required to induce pyruvate kinase M synthesis and this function can be uncoupled from other aspects of cellular differentiation. The uncoupling of pyruvate kinase maturation from cellular differentiation and the observation of only a single, 2.3 kilobase, pyruvate kinase mRNA molecule at different stages of the postnatal development of rat brain support the concept that the K- to M-isoform transformation is a post-transcriptional event. The effect of the individual components of this chemically defined medium on pyruvate kinase specific activity was studied by eliminating one component at a time. The increase in activity was found to be completely dependent upon fibroblastic growth factor and prostaglandin F2 alpha and was partially dependent on the simultaneous presence of insulin.

Aging↗
PubMed1977

Purification and molecular properties of the AMP-activated pyruvate kinase from Escherichia coli.

The AMP-activated pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Escherichia coli has been purified 200 times through a three-step procedure which gives a homogeneous preparation with a specific activity of 110. The enzyme appears to be a tetramer of molecular weight 190 000. Subunits (molecular weight 51 000) show a single amino-terminal amino acid (serine) and appear as a single band in polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The enzyme crystallizes in conditions of reduced dielectric constant of the solvent in the pH range 6.5-7.5. Kinetic and regulatory properties of the purified enzyme are similar to those described for crude preparations of the enzyme.

Amino Acids↗
PubMed1986

Phosphorylation of liver pyruvate kinase by Ca++/calmodulin-dependent protein kinase: characterization of two phosphorylation sites.

Rat liver pyruvate kinase is phosphorylated by calcium/calmodulin-dependent protein kinase II at serine and threonine residues in a 3-4 kDa CNBr fragment located near the amino terminus. The two sites of phosphorylation were separated by reverse-phase HPLC of a thermolysin digest. Sequence analysis established the sites of phosphorylation as follows: Leu-Arg-Arg-Ala-Ser(PO4)-Val-Ala-Gln-Leu-Thr(PO4)-Gln-Glu.

Amino Acid Sequence↗
PubMed1987

Purification and kinetic properties of pyruvate kinase isoenzymes of Salmonella typhimurium.

Two forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) present in Salmonella typhimurium were purified to homogeneity from the same cultures by (NH4)2SO4 fractionation and gel filtration, anion-exchange and affinity chromatography. Mr values, subunit structure, amino acid composition and activity and stability conditions were determined for the two forms. Kinetic and regulatory properties of the two purified isoenzymes were studied.

Adenine Nucleotides↗
PubMed1984

Pyruvate kinase from Aspergillus niger: a regulatory enzyme in glycolysis?

Pyruvate kinase from the filamentous, citric acid producing fungus Aspergillus niger was purified about 100-fold by ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration. The addition of fructose-1,6-diphosphate was necessary to prevent loss of activity during purification. The enzyme purified in the presence of fructose-1,6-diphosphate (FDP) exhibits hyperbolic kinetics with respect to phosphoenolpyruvate (PEP) and ADP. Monovalent cations activated the enzyme (K+, NH4+). FDP neither activated nor inhibited the enzymatic activity from extracts freshly prepared in the absence of exogenous FDP; ATP showed a weak activation. In contrast the enzyme from crude extracts which had been stored in the presence of glycerol for 3 days showed activation by FDP or a metabolite thereof and inhibition by ATP. In the absence of FDP sigmoidal kinetics were obtained with respect to PEP, which became hyperbolic kinetics after addition of FDP. ATP inhibition turned into slight ATP activation in the presence of FDP. However, it was possible to reactivate inactivated pyruvate kinase (after dialysis in the absence of FDP) by adding FDP to the enzyme assay. From these results and because of the very high affinity of pyruvate kinase for FDP (Ka less than 0.1 microM), it is concluded that the enzyme probably has FDP bound to the protein in vivo. The significance of this hypothesis to the regulation of glycolysis in A. niger, with special reference to the mechanism of citric acid accumulation, is discussed.

Adenosine Diphosphate↗
PubMed1990

Multiple copies of the pyruvate kinase gene affect yeast cell growth.

The Saccharomyces cerevisiae pyruvate kinase gene (PYK1) was transformed into yeast using the multicopy vector pJDB207. Growth rates and PYK1 gene expression levels varied considerably amongst the transformants. Yeast transformants expressing the PYK1 gene at high levels formed small colonies compared with those expressing the gene at relatively low levels. Slow-growing transformants 'reverted' at high frequency to more rapid growth, and this correlated with decreases in PYK1 gene copy number and PYK1 mRNA abundance. This apparent selection against PYK1 over-expression was disrupted by the introduction of a stop codon at the 5'-end of the PYK1 coding region, thus confirming that the growth effects were mediated by the PYK1 gene. However, massive overproduction of pyruvate kinase in yeast, using multiple copies of a PGK:PYK gene fusion, had no significant effect upon cell growth. This suggests that the deleterious effect upon the host yeast cell is mediated by abnormally high levels of the wild-type gene or PYK1 mRNA, rather than by increased pyruvate kinase levels.

Base Sequence↗