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At least 127 records · Page 7Linked to original sources
PubMed1975

Affinity elution of pyruvate kinase from phosphocellulose.

Pyruvate kinase from ascites tumour cells can be eluted from phosphocellulose by very low concentrations of phosphoenolpyruvate, fructose 1,6-bisphosphate, adenosine 5'-diphosphate and pyrophosphate, respectively. The appropriate limiting conditions for "facilitated desorption" of the enzyme from phosphocellulose by these ligands have been elaborated for achieving maximum selectivity and recovery in the process of its purification. This method has been designated as "affinity elution chromatography" owing to the specific interactions between a ligand as a constituent of the eluting medium with the adsorbed enzyme, which causes its selective desorption from the ion-exchanger. Affinity elution with phosphoenolpyruvate has been found to be very effective for preparation of the M-types of pyruvate kinase. A specific activity of 420 for an almost homogeneous preparation of pyruvate kinase from ascites tumour cells has maximally been obtained.

Animals↗
PubMed1977

Characterization of pyruvate kinase from the liver of a patient with aberrant erythrocyte pyruvate kinase, PK Nagasaki.

The characterization of the L-type PK were made of PK extracted from the liver of a patient with congenital hemolytic anemia associated with an erythrocyte PK variant, PK Nagasaki. The L-type PK of PK Nagasaki showed the following parameters: slow migration on electrophoresis, high Km for PEP without F-1,6-P2, less activation by F-1,6-P2, normal Km for ADP, high utilization of UDP, acidic pH optimum, and instability to urea and heat. These tests served to differentiate this L-type PK variant from the other variants previously reported. At the same time, both the Km for PEP with F-1,6-P2 saturation and the electrophoretic mobility of L-type PK were found to be different from those of the erythrocyte PK and PK Nagasaki. Though the liver cell, with regard to L-type PK, has only the less functional and less stable mutant L-type PK there is no evidence of liver dysfunction or damage, although there is chronic hemolytic anemia.

Adolescent↗
PubMed1996

Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

Acid Phosphatase↗
PubMed1985

Purification and properties of rat brain pyruvate kinase.

Rat brain pyruvate kinase was purified to near homogeneity by a three-step process involving ammonium sulfate precipitation and phosphocellulose and Blue-Sepharose CL-6B column chromatography. The enzyme migrated on polyacrylamide gel along with a commercial sample of rabbit muscle pyruvate kinase. The enzyme showed a hyperbolic relationship with phosphoenolpyruvate and ADP, with apparent Km's of 0.18 and 0.42 X 10(-3) M, respectively. The enzyme was inhibited by ATP, the effect being more pronounced at unsaturating concentrations of phosphoenolpyruvate. L-Phenylalanine was found to be a strong inhibitor of the enzyme, with the Ki for inhibitor being 0.11 mM. The inhibition by phenylalanine was more pronounced at pH 7.4 than at pH 7.0, and appeared to be competitive with phosphoenolpyruvate. L-Alanine and fructose 1,6-bisphosphate prevented the inhibition of the enzyme by phenylalanine. Ca2+ was found to be a strong inhibitor of the enzyme, and the inhibition was more marked at saturating phosphoenolpyruvate concentrations. The kinetic properties of the purified brain pyruvate kinase suggest that the enzyme may be distinct from the muscle or liver enzymes.

Adenosine Monophosphate↗
PubMed1987

Isolation and sequence determination of an active site peptide of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was inactivated by 2', 3'-dialdehyde ADP with the incorporation of one molecule of reagent per enzyme subunit. The inactivated protein was digested with trypsin after reduction and carboxymethylation. The labeled peptide was isolated by gel filtration and further purified by HPLC. The peptide was sequenced both by liquid-phase and gas-phase automatic Edman degradation. A 34-residue peptide was obtained. This peptide is identical to a tryptic peptide labeled with trinitrobenzenesulfonate, isolated and sequenced by Johnson et al. (Biochem. Biophys. Res. Commun. (1979) 90, 525-530) from bovine muscle pyruvate kinase. Available evidence suggests that dialdehyde ADP labels the enzyme at the same lysine in position 25 of the peptide, as found by Johnson et al. The high homology between the isolated peptide and regions of other pyruvate kinases from low to high eukaryotes supports the idea that this peptide is related to the enzyme active site.

Adenosine Diphosphate↗
PubMed1978

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase.

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.

Animals↗
PubMed1988

ADP-ribosylation suppresses phosphorylation of the L-type pyruvate kinase.

L-type pyruvate kinase (EC 2.7.1.40) purified from pig liver was ADP-ribosylated by incubation with NAD and ADP-ribosyltransferase purified from hen liver nuclei. Maximal incorporation of the ADP-ribose moiety from NAD into the L-type pyruvate kinase was 0.98 mol/mol of subunit. The Km values for NAD and L-type pyruvate kinase were 0.17 mM and 9.7 microM, respectively. ADP-ribosylation of the L-type pyruvate kinase resulted in suppression of the subsequent phosphorylation catalyzed by cAMP-dependent protein kinase. The ADP-ribosylation-induced suppression of phosphorylation of the L-type pyruvate kinase also resulted in suppression of the phosphorylation-induced inactivation. Amino acid analysis, after exhaustive sequential digestion of ADP-ribosyl-L-type pyruvate kinase with pepsin, aminopeptidase M and carboxy-peptidase B showed arginine to be the ADP-ribose-accepting amino acid. These results together with finding of the ADP-ribosyltransferase activity in mammalian liver cytosol (Moss, J. and Stanley, S.J. (1981) J. Biol. Chem. 256, 7830-7833) suggest that ADP-ribosylation may participate in the regulation of the L-type pyruvate kinase activity through changes in the rate of phosphorylation.

Adenosine Diphosphate Ribose↗
PubMed1984

A rapid purification method for human erythrocyte pyruvate kinase.

Human erythrocyte pyruvate kinase (ATP: pyruvate phosphotransferase, E.C.2.7.1.40) is purified 30,000-fold, using a method which includes ammonium sulfate precipitation, Sephadex G-75 filtration, and Blue Dextran-Sepharose 4B chromatography. The enzyme is resolved into two peaks on Blue Dextran-Sepharose 4B. The first peak with sp act of 300 corresponds to the mature form (R4) whereas the second peak with sp act of 180 corresponds to R2R'2. Peaks I and II give one band on 10% polyacrylamide gel without SDS. Peak II gives two bands on 10% SDS gel with molecular weights 60,000 (R') and 57,500 (R). On the other hand, peak I gives only one band on 10% SDS gel having a molecular weight of 57,500. Both the R4 and R2R'2 forms of the enzyme have the same pH optimum of 7.2.

Chemical Phenomena↗
PubMed1991

Purification and kinetic properties of pyruvate kinase from Brochothrix thermosphacta.

Pyravate kinase (ATP: pyruvate 2-0 phosphotransferase E.C.2.7.1.40) was purified from Brochothrix thermosphacta. The enzyme is a homotetramer of monomer Mr 58,000. Fructose-1,6-bisphosphate stimulates activity and promotes hyperbolic kinetics although it is not essential for enzyme activity. The positive effect of fructose-1,6-bisphosphate on activity is repressed by inorganic phosphate which enhances cooperative kinetics. Unlike pyruvate kinases from other sources, the Brochothrix enzyme is uncompetitively inhibited by glucose-6-phosphate, although at high concentration. ATP is a strong inhibitor of pyruvate kinase and shifts the residual activity/pH profile towards more alkaline values.

Adenosine Diphosphate↗
PubMed1981

Immunofluorescence and histochemical methods for neural M1 pyruvate kinase localization.

The distribution of pyruvate kinase (ATP pyruvate phosphotransferase, EC 2.7.1.40) in the nervous system has been studied by both immunofluorescence and a histochemical procedure using nitro blue tetrazolium. The localization in various parts of rat central nervous system in situ, cerebellar and cerebral cortex, was compared to that found in vitro in cultures of cerebellum, spinal ganglia, cerebral astrocytes, and skin fibroblasts. (1) Pyruvate kinase was found predominantly in the cytoplasm of neuronal cell bodies. (2) Large neurons were better visualized than small ones. (3) No glial localization was clearly demonstrated in situ, although this does not rule out the presence of some M1 pyruvate kinase. (4) Regions expected to be rich in nerve terminals, such as the cerebellar glomeruli or the cerebellar molecular layer, showed intense staining even when the cell bodies themselves were negative. This was expected, owing to the previous demonstration of the presence of M1 pyruvate kinase in nerve ending by subcellular fractionation methods. (5) The localization was similar in situ and in tissue culture, except that nerve processes were better seen in the latter and astrocytes were sometimes stained in vitro. (6) Variation in intensity of staining was observed in similar cell types in the same section or in the same culture. This could represent different metabolic or functional or maturational states.

Animals↗
PubMed1976

Defective erythrocyte pyruvate kinase.

A defective pyruvate kinase (EC 2.7.1.40) is described. The abnormal PK is characterized by a shift in the R in equilibrium T equilibrium to the T-state. The Ko.5 for the substrate phosphoenol pyruvate is about 6 times higher than for the normal enzyme, while the KM value for the positive effector Fru-1, 6-P2 is increased. In agreement with a shift to the T-state is the increased affinity of the abnormal enzyme for the negative effectors ATP and alanine. The results are discussed in relation to other abnormal pyruvate kinases.

Adenosine Triphosphate↗
PubMed1975

Phosphoenolpyruvate hydrolase activity of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase catalyzes the hydrolysis of P-enolpyruvate at the same active site which catalyzes the physiologically important kinase reaction. The hydrolase activity is lower than the kinase activity by a factor of at least 10(3). There are specific monovalent cation and divalent cation requirements. No other cofactors are required. The relative activation of the pyruvate kinase for the hydrolase reaction is: Ni(II) greater than Co(II) greater than Mg(II) greater than Mn(II). This parallels the rates of nonenzymatic hydrolysis of P-enolpyruvate (Benkovic, S.J., and Schray, K.J. (1968) Biochemistry 7, 4097-4102). The pH rate profiles of the hydrolase and kinase reactions activated by Ni(II) and Co(II) are similar, suggesting common features in their mechanisms. In contrast to the kinase reaction, the reaction velocity of the hydrolase increases at high Co(II) concentrations indicating a second mode for hydrolysis.

Animals↗
PubMed1983

Purification and properties of pyruvate kinase from Mycobacterium smegmatis.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S0.5v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest Km value. The enzyme showed an absolute requirement for divalent cations (either Mg2+ or Mn2+), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg2+-activated enzyme to varying degrees (Ni2+ greater than Zn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+). The differences in the kinetic responses of the enzyme to Mg2+ and Mn2+ are discussed.

Adenosine Triphosphate↗
PubMed1985

Comparative studies on soluble and immobilized rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase was immobilized by covalent attachment to a polyacrylamide support (Akrilex C) containing carboxylic functional groups. As a result of immobilization, the pH optimum for catalytic activity shifted into a more alkaline direction. The apparent Km value with phosphoenolpyruvate increased, and that with ADP slightly decreased. With respect to the stability against urea and thermal inactivation, the immobilized pyruvate kinase seemed to be the more stable at lower urea concentrations and between 45 and 55 degrees C. At 1.5 and 2.5M urea and at higher temperature, there were no marked differences between the soluble and the immobilized enzyme.

Animals↗
PubMed1998

Cooperativity in Bacillus stearothermophilus pyruvate kinase.

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose- 5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.

Amino Acid Sequence↗
PubMed1975

Subunit structure and some properties of pyruvate kinase of Neurospora.

Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000-52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS-polyacrylamide gels. The enzyme has an isoelectric point of 6.35-6.41; Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg-2+, is required for activity. At low concentrations, Ca-2+ acts as an activator of pyruvate kinase but it is inhibitory at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.

Allosteric Regulation↗
PubMed1976

Purification and properties of pyruvate kinase from Streptococcus lactis.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) of Streptococcus lactis C10 is activated by fructose 1,6-diphosphate (Fru-1,6-P2), activity being a sigmoidal function of activator concentration. The FDP0.5V (Fru-1,6-P2 concentration giving half-maximal velocity) is markedly increased in the presence of low concentrations of inorganic phosphate; 1 mM phosphate increases the FDP0.5V value 6-fold. Although the intracellular level of Fru-1,6-P2 (12-18 mM) in exponentially growing cells on the medium used is much greater than the FDP0.5V for pyruvate kinase (0.2 mM) as determined in triethanolamine-HCl buffer, a much higher Fru-1,6-P2 concentration may be required to activate the enzyme in vivo to overcome phosphate inhibition. Tris and maleate also inhibit the enzyme. At low concentrations of Fru-1,6-P2 (0.1 mM), reaction rate is a sigmoidal function of both phosphoenolpyruvate and adenosine diphosphate (ADP) concentrations; at near saturating concentrations of activator (1 mM) the response to varying ADP is hyperbolic while the response to varying phosphoenolpyruvate becomes much less sigmoidal. The affinity for both substrates (especially phosphoenolpyruvate) is also increased by increasing the concentration of Fru-1,6-P2. The affinity of the enzyme for guanosine disphosphate (GDP) is 12-13 times that for ADP under the assay conditions used. The Streptococcus lactis pyruvate kinase has a molecular weight of 240000 with a subunit molecular weight of 60000.

Adenosine Diphosphate↗