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PubMed1995

Purification and properties of the pyruvate kinase isozyme M1 from the pig brain.

There are four pyruvate kinase isozymes in vertebrate tissues, designated as L, M1, M2, and R. Although pyruvate kinases have been purified and characterized from pig liver, muscle, kidney, and heart, the brain isozyme has not. The aim of this work was to purify, characterize and make an isozymic designation for the pig brain pyruvate kinase. Purification was accomplished by chromatography on phosphocellulose, Sephadex G200, and blue-dextran agarose columns. The molecular weight of the native enzyme was determined by sucrose density centrifugation. The degree of purity, and subunit molecular weight were determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was estimated by the rapid isoelectric focusing method in sucrose gradients. The pH optimum, and kinetics in the presence and absence of fructose-1,6-diphosphate were determined spectrophotometrically. The purification scheme used resulted in a 382-fold purification of pig brain pyruvate kinase, and a final specific activity of 191 Units/mg protein. As estimated by scanning of the sodium dodecyl sulfate polyacrylamide gels, the purification scheme also resulted in a preparation that was of at least 98% purity. Pig brain pyruvate kinase has a native molecular weight of approx. 230,000, and a subunit molecular weight of approx. 60,000. The pI was determined to be approximately 8.0, while the pH optimum was estimated at pH 7.4. Fructose-1,6-diphosphate had no effect on either the Km for phospho(enol)pyruvate, or the Vmax of the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

The long-term effect of glucagon on pyruvate kinase activity in primary cultures of hepatocytes.

Glucagon caused a marked decrease in the total L-pyruvate kinase activity of control hepatocytes maintained in monolayer culture (t1/2 = 54 h), while the addition of insulin to hepatocytes isolated from a fasted rat caused a four- to fivefold increase in the total enzyme activity. Maintenance of L-pyruvate kinase in control cultures of hepatocytes was shown to require insulin. However, when 1 microM glucagon was present in the medium, the total L-pyruvate kinase activity was not maintained even in the presence of 1 microM insulin, but rather the total L-pyruvate kinase activity of the cells steadily declined from 12.1 to 5.7 units/mg DNA by the 6th day in culture. The increase in the total L-pyruvate kinase activity of fasted hepatocytes cultured in the presence of insulin was shown to result from an increase in protein synthesis, since actinomycin D and cycloheximide blocked the insulin-induced increase in the enzyme activity. The addition of 1 microM glucagon to cultures of fasted hepatocytes also blocked the insulin-induced increase in total L-pyruvate kinase activity. Since glucagon decreased the total L-pyruvate kinase activity in control hepatocytes and blocked the increase in L-pyruvate kinase activity in fasted hepatocytes, it is suggested that, in addition to the phosphorylation of L-pyruvate kinase by a cAMP-dependent protein kinase, glucagon also acts to decrease the synthesis of L-pyruvate kinase in vitro.

Animals↗
PubMed1979

The effects of season and temperature on D-lactate dehydrogenase, pyruvate kinase and arginine kinase in the foot of Helix pomatia L.

The effects of pH, season, environmental and experimental temperatures on the activities and kinetic parameters of D-lactate dehydrogenase, pyruvate kinase and arginine kinase from the foot of the pulmonate snail Helix pomatia were analyzed. Both in phosphate and Tris buffers D-lactate dehydrogenase was the enzyme with the most acid maximum, arginine kinase that with the most alkaline, whilst pyruvate kinase occupied an intermediate position. Pyruvate kinase activity, measured at 20 degrees C, was positively correlated with the environmental temperature at the moment of collecting the animal, whereas neither arginine kinase nor D-lactate dehydrogenase showed such a relationship. A seasonal study based on approximately 100 specimens established that arginine kinase activity remained the same throughout the year. Pyruvate kinase activity was slightly lower, and D-lactate dehydrogenase activity significantly higher, in winter than in summer animals. Snails subjected in spring to a short warm-up period before enzyme extraction showed extreme variability and some extraordinarily high values of pyruvate kinase activity, suggesting that either season or elevated temperature may have an immediate effect on the activity of this enzyme. Individual variability of all three enzymes ranges from 300 to 400%. The activities of pyruvate kinase and D-lactate dehydrogenase are strongly correlated in summer, forming a "constant-proportion-group", whereas in winter, with D-lactate dehydrogenase activity increasing and pyruvate kinase activity decreasing these two enzymes become "uncoupled". The Km value of pyruvate kinase is independent of experimental temperature between 10 and 25 degrees C, whereas that of D-lactate dehydrogenase and arginine kinase increases about three-fold within this range. Thus the temperature relationship of a single enzymic reaction cannot be used as an arguemnt for or against the occurrence of temperature compensation of whole animal metabolism. The possibility of modulation of enzyme activity by environmental temperature is discussed.

Acclimatization↗
PubMed1990

Purification and properties of pyruvate kinase from Dictyostelium discoideum.

Pyruvate kinase (EC 2.7.1.40) from aggregating Dictyostelium discoideum cells has been purified to homogeneity. It has a monomeric molecular weight of 66kD and is tetrameric in low ionic strength buffers. The enzyme is not regulated by fructose 1,6-bisphosphate or by alanine and appears to resemble the M1 isoenzyme from rat liver most closely, although its activity is not inhibited by ATP.

Dictyostelium↗
PubMed1986

Purification and properties of pyruvate kinase from Bacillus stearothermophilus.

Pyruvate kinase was purified to homogeneity from a moderate thermophile, Bacillus stearothermophilus. The molecular weight of the enzyme was found to be 250,000 on gel filtration and 242,000 on sedimentation analysis. The enzyme consisted of four identical subunits of a molecular weight of 62,000-64,000. There were no remarkable differences between the thermophilic enzyme and mesophilic enzymes in amino acid composition, secondary structure, mono- and di-valent cation requirements for activity or specificity for nucleoside diphosphates. But the thermophilic enzyme was stable at high temperature and for a longer period of storage at lower temperature. Its specific activity was relatively high even at a low temperature (30 degrees C). The enzyme exhibited homotropic positive cooperativity for phosphoenol-pyruvate, but not for ADP. It was allosterically activated by AMP, ribose 5-phosphate and nucleoside monophosphates, but not by fructose 1,6-bisphosphate. Activation by AMP and ribose 5-phosphate, and inhibition by inorganic phosphate were also observed even at the physiological temperature (60 degrees C) for the thermophile.

Adenosine Diphosphate↗
PubMed1988

Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans.

The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI+. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration--55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration. A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth ona glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region. However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.

Aspergillus nidulans↗
PubMed1976

Comparative kinetic studies on the L-type pyruvate kinase from rat liver and the enzyme phosphorylated by cyclic 3', 5'-AMP-stimulated protein kinase.

The kinetics of rat liver L-type pyruvate kinase (EC 2.7.1.40), phosphorylated with cyclic AMP-stimulated protein kinase from the same source, and the unphosphorylated enzyme have been compared. The effects of pH and various concentrations of substrates, Mg2+, K+ and modifiers were studied. In the absence of fructose 1, 6-diphosphate at pH 7.3, the phosphorylated pyruvate kinase appeared to have a lower affinity for phosphoenolpyruvate (K0.5=0.8 mM) than the unphosphorylated enzyme (K0.5=0.3 mM). The enzyme activity vs. phosphoenolpyruvate concentration curve was more sigmoidal for the phosphorylated enzyme with a Hill coefficient of 2.6 compared to 1.6 for the unphosphorylated enzyme. Fructose 1, 6-diphosphate increased the apparent affinity of both enzyme forms for phosphoenolpyruvate. At saturating concentrations of this activator, the kinetics of both enzyme forms were transformed to approximately the same hyperbolic curve, with a Hill coefficient of 1.0 and K0.5 of about 0.04 mM for phosphoenolpyruvate. The apparent affinity of the enzyme for fructose 1, 6-diphosphate was high at 0.2 mM phosphoenolpyruvate with a K0.5=0.06 muM for the unphosphorylated pyruvate kinase and 0.13 muM for the phosphorylated enzyme. However, in the presence of 0.5 mM alanine plus 1.5 mM ATP, a higher fructose 1, 6-diphosphate concentration was needed for activation, with K0.5 of 0.4 muM for the unphosphorylated enzyme and of 1.4 muM for the phosphorylated enzyme. The results obtained strongly indicate that phosphorylation of pyruvate kinase may also inhibit the enzyme in vivo. Such an inhibition should be important during gluconeogenesis.

Adenosine Diphosphate↗
PubMed2005

Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus.

Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6(2)22, with unit-cell parameters a = b = 145.97, c = 118.03 A.

Crystallization↗
PubMed1977

[Energy metabolism of erythrocytes in pyruvate kinase enzymopathies].

Until now pyruvate kinase enzymopathies have been described only for red blood cells. On the basis of these results special structural properties of the erythrocyte PK was assumed, which are not yet totally established. PK defects may cause a nonspherocytic hemolytic anemia. This enzymopathy is characterized by a polymorphism, which is expressed in more than 5 different pathological variants. Up to now 16 cases of PK deficiency have ben diagnozed in the GDR. The following parameters are used for the characterization of the PK: the PEP-dependance, the inhibition by ATP and alanine, the specificity to nucleotides, the stability to temperature and urea and the maturation dependence. Two pathological variants of the PK with a decreased PEP-affinity are described. Furthermore the differences in the energy metabolism of the red blood cells of these two patients under aerobic and anaerobic conditions are discussed.

Adenosine Triphosphate↗
PubMed1984

The regulatory properties of yeast pyruvate kinase.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 where the relationships between the initial velocities of the catalysed reaction and the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ are non-hyperbolic. The findings were represented empirically by the exponential model for a regulatory enzyme. The analysis shows that ADP, phosphoenolpyruvate and Mg2+ display positive homotropic interaction in their binding behaviour with (calculated) Hill slopes at half-saturation equal to 1.06, 2.35 and 3.11 respectively [Ainsworth (1977) J. Theor. Biol. 68, 391-413]. The direct heterotropic interaction between ADP and phosphoenolpyruvate is small and negative, but the overall interaction between these substrates becomes positive when their positive interactions with Mg2+ are taken into account. The heterotropic interactions of the substrates, though smaller in magnitude, are comparable with those revealed by the rabbit muscle enzyme [Ainsworth, Kinderlerer & Gregory (1983) Biochem. J. 209, 401-411], and it is suggested that they have a common origin in charge interactions within the active site.

Adenosine Diphosphate↗
PubMed1995

Genetic test for pyruvate kinase deficiency of Basenjis.

Pyruvate kinase (PK) deficiency is an autosomal, recessive, inherited disease of Basenjis that causes chronic, regenerative, hemolytic anemia. Diagnostic methods currently used to identify carrier animals rely on measurement of erythrocyte PK activity and frequently give equivocal results. A genetic test incorporating polymerase chain reaction amplification of genomic DNA and restriction fragment length polymorphism has been developed to determine the PK genotype of Basenjis. To determine whether results of this genetic test compared with results of standard tests for PK deficiency, erythrocyte PK activity, hematocrit, and reticulocyte counts were determined in, and the genetic test was performed on, 24 dogs. The genetic test accurately identified the 11 dogs whose PK genotype was known prior to this study, and results were consistent with results of measuring erythrocyte PK activity in the remaining 13 dogs. The genetic test may be of value in determining PK genotype of Basenjis.

Anemia, Hemolytic↗
PubMed1989

Purification and regulatory properties of pigeon erythrocyte pyruvate kinase.

1. Pigeon erythrocyte pyruvate kinase (PK) was purified 22,000 fold by successive column chromatography on Sephadex DEAE A-50 and Red Agarose. The resulting enzyme preparation had a specific activity of 815.3 U/mg protein and an overall yield of 18.5%. 2. The molecular weight, as determined by gel filtration on Sephadex G-200 was 152,000. 3. Isoelectric focusing in the pH range of 3-10 showed that pigeon erythrocyte contained at least 3 PK isozymes with isoelectric points of 5, 5.7 and 6. 4. The variation of activity of PK at various ADP and phosphoenolpyruvate (PEP) concentrations was studied. The Km values for ADP and PEP were 0.40 and 0.46 mM respectively. 5. The enzyme was activated by FDP, and inhibited by ATP, highly phosphorylated inositol derivatives and 2,3-DPG: 6. It was activated by K+ and Mg2+ ions. 7. Phosphorylated hexoses and Pi stimulated the activity of PK. 8. The regulatory role of PK of pigeon erythrocytes, which lack the typical 2,3-DPG bypass, is discussed.

Animals↗
PubMed1988

Assay of rat plasma pyruvate kinase activity with luciferin-luciferase.

An assay method for pyruvate kinase in rat plasma is described. Plasma samples were incubated with ADP and phosphoenolpyruvate in Tris buffer solution. The ATP produced by pyruvate kinase was measured by photocounting after the addition of a commercially available luciferin-luciferase preparation. Interference by ATP or adenylate kinase originally present in the sample was removed by a high degree of dilution. The assay is sensitive, reproducible, and rapid, especially when used for large numbers of samples. By this method, pyruvate kinase activity in normal rats was determined to be 0.51 +/- 0.05 (n = 6) U/ml plasma. In rats fed a vitamin E-deficient basal diet for 7, 10, or 14 weeks, pyruvate kinase activities were 0.70 +/- 0.11, 1.64 +/- 0.51, and 4.28 +/- 0.85 (n = 6) U/ml plasma, respectively. This method appears to be useful for the determination of pyruvate kinase activity in nutritional or pharmacological studies.

Animals↗
PubMed1988

Expression of L- and M-type pyruvate kinase in human tissues.

Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes of PK L and M. Differential splicing produces L- and R-type PK mRNA and M1- and M2-type PK mRNA from the PK L gene and the PK M gene, respectively. The nucleotide sequences of the 3'-noncoding region are the same between the L- and the R-type PK and between the M1- and the M2-type PK. We isolated 3'-noncoding sequences of human L- and M2-type PK cDNA to construct L-type and M-type PK specific probes. With these probes, we performed Northern blot analysis of the RNA samples extracted from human tissues. Northern blot analysis showed that both kidney and liver had mRNAs that hybridized with both the L and M probes. Small intestine, skeletal muscle, brain, testis, and lung mRNAs hybridized only with the M probe. Our probes are considered useful for the detection of the types of PK isozymes expressed in small amounts, which are very difficult to detect using the conventional PK polyacrylamide gel electrophoretic method.

DNA Restriction Enzymes↗
PubMed1980

Analysis of the renaturation kinetics of bovine muscle pyruvate kinase.

Bovine type M pyruvate kinase can be reversibly denatured by solutions of guanidine-HCl. Subsequent dilution of the enzyme into buffer containing 2-mercaptoethanol or dithiothreitol results in recovery of enzymatic activity with half-times that vary from 185 min at 0 degrees C to 4 min at 45 degrees C. In the temperature range 0-25 degrees C, 90% of the enzymatic activity is recovered. Above about 32 degrees C, the recovery drops off sharply, wih a yield of only 13% at 45 degrees C. Removal of inactive nonspecific aggregates and denatured monomer by gel filtration yields an enzyme with the same specific activity as the starting material. At enzyme concentrations below 3 microns/mL at 16 degrees C or below 25 micron/mL at 7.8 degrees C, the reactivation kinetics show a concentration dependence. At higher concentrations of protein and at temperatures of 16 degrees C or higher, no protein concentration dependence is seen, and the rate of reactivation is described by two first-order relaxations. The rate constants have apparent activation energies of 10.6 and 11.9 kcal/mol. Combinding the results presented here with earlier work from this laboratory [Cardenas, J. M., & Dyson, R. D. (1973) J. Biol. Chem. 248, 6938-6944; Cardenas, J. M., Hubbard, D. R., & Anderson, S. (1977) Biochemistry 16, 191-197] leads to the conclusion that a rapid, major folding produces two species which undergo transconformational steps. This is followed by subunit association which yields the native tetramer.

Animals↗
PubMed2006

Differentiating a ligand's chemical requirements for allosteric interactions from those for protein binding. Phenylalanine inhibition of pyruvate kinase.

The isoform of pyruvate kinase from brain and muscle of mammals (M(1)-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the l-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M(1)-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the beta-carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the beta-carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 A structure of M(1)-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the l-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.

Alanine↗
PubMed1975

Regulation of C3-enzymes in facultative phototrophic bacteria: the cold-labile pyruvate kinase of Rhodopseudomonas sphaeroides.

Pyruvate kinase (EC2.7.1.40) from Rhodopseudomonas sphaeroides was purified 40-fold be precipitation with protamine sulfate and ammonium sulfate followed by gel-filtration. The preparations obtained from cells grown with different carbon sources or cultural conditions differ with respect to specific activity but not with respect to molecular weight (250000 dalton) or regulatory properties. The phosphoenolpyruvate (PEP)-saturation cruve of the enzyme is sigmoidal with Hill coefficients varying from nH equals 1.8 (pH 9.2) to 2.7 (pH 6.0). The enzyme is activated by adenosinemonophosphate (AMP) and the sugarmonophosphates ribose-5-phosphate (R-5-P), glucose-6-phosphate (G-6-P), and-to a lesser extent-fructose-6-phosphate (F-6-P). Fructose-1.6-bisphosphate (FDP) has no measurable effect. Inhibitors of the enzyme are adenosinetriphosphate (ATP), inorganic phosphate (Pi) and the dicarboxylic acids succinate and fumarate. Kinetic analysis reveals that the sugar-phosphates and the dicarboxylic acids act as true allosteric ligands, whereas the effects of AMP, ATP, and Pi cannot be interpreted soley in terms of allosteric interactions. Cold-treatment of the enzymes lead to a rapid loss of activity, but does not change the regulatory properties of the enzyme. Analysis of the kinetics of cold-inactivation and its reversal at 30 percent C, together with studies on the gelfiltration behaviour of the native and the cold-treated enzyme make it likely that the cold-induced loss of activity is due to a dissociation of the enzyme.

Adenosine Monophosphate↗
PubMed1974

Kinetic properties of cerebral pyruvate kinase.

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP(2-) was competitive for MgADP(-) but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP(2-) with respect to both substrates. The K(i)(MgATP(2-)) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1-2mm) concentrations of MgATP(2-). Substrate kinetic constants were K(m) (MgADP(-))=0.47mm, K(m) (phosphoenolpyruvate)=0.08mm. Free Mg(2+) at very high concentrations (over 10mm) was inhibitory (K(i)=20-32mm). Neither ADP(3-) nor 5'-AMP(2-) inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.

Adenosine Diphosphate↗