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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The steady-state kinetics of human skeletal muscle pyruvate kinase (MA) and its RNA-complex (MB) has been examined and compared. Kinetic studies revealed significant differences in kinetic properties with respect to free and complex form of pyruvate kinase. The MA form follows a simple Michaelis-Menten kinetics in contrast with the MB form, which displays a negative cooperativity with respect to ADP. Vmax for the complex is 40-60% that for free enzyme. Heterologous RNA is a noncompetitive inhibitor of free enzyme but the kinetics of the complex (MB) is not affected. In presence of 1.0 mM ATP in an assay mixture the kinetic constants of the complex were unchanged except for Vmax, which increased by nearly 60%. Aged preparations of free enzyme (MA) were activated by 100% and more, but the native enzyme was inhibited by 22%. Inorganic phosphate is a potent activator of both forms of pyruvate kinase. In presence of 50 mM K-phosphate the apparent Michaelis constant and interaction coefficient are unchanged, but Vmax for free enzyme increases by 35% and for the complex by 70%, respectively. The specific activity of aged MA form can be restored to the original value after incubation of the enzyme in 50 mM K-phosphate, pH 7.6, or by addition of ATP (1.0 mM) to the assay mixture.
Mol Cell Biochem 1982 Jun 11
PMID:Kinetic properties of human muscle pyruvate kinase. 711 Jan 27

The influence of dexamethasone on the isozyme patterns of ATP-hexose phosphotransferases, aldolase and pyruvate kinase of adult rat hepatocytes maintained in primary cultures has been studied. A progressive loss of the typical adult liver isozymes glucokinase, pyruvate kinase L and aldolase B, with a simultaneous increase of both pyruvate kinase A and hexokinase activities, was observed in hepatocytes cultured in the absence of added glucocorticoid. When the culture medium was supplemented with 10(-7)M dexamethasone, the adult liver patterns of pyruvate kinase and aldolase were preserved for at least seven days of culture, the initial level of glucokinase was maintained for three days, and the rise of hexokinase activity was delayed and partially blocked. These results are discussed in relation to the known beneficial effect of glucocorticoids on the survival of cultured hepatocytes.
Mol Cell Biochem 1982 Jun 11
PMID:Effect of dexamethasone on the isozyme pattern of adult rat liver parenchymal cells in primary cultures. 711 Jan 29

The structural transitions of the tetrameric rabbit muscle pyruvate kinase induced by guanidine hydrochloride and urea are characterized by elastic and quasi-elastic light-scattering, sedimentation velocity, and intrinsic viscosity experiments as well as by protein fluorescence, circular dichroism, and enzymic activity measurements. The transition curves are shown to be reversible. We find a new pathway of unfolding which is different from that described in the literature: The first intermediate with increasing concentration of denaturant is a less compact and inactive tetramer which can be renatured if substrates are added. Dissociation of the tetramer results in an expanded dimer with a partial loss of the secondary structure. The final state is a completely disordered monomer. These intermediates are consistent with a domain structure of pyruvate kinase, as it was suggested by Stammers & Muirhead [Stammers, D. K., & Muirhead, H. (1975) J. Mol. Biol. 95, 213--225] on the basis of their X-ray data. Using Schellman's solvent denaturation model [Schellman, J. A. (1978) Biopolymers 17, 1305--1322], we calculate the free energies of stabilization of the folding--unfolding equilibrium.
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PMID:Reversible solvent denaturation of rabbit muscle pyruvate kinase. 721 11

The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The Km for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a pKa of 6.5-6.7. The cooperative interaction of the multiple phosphoenolpyruvate (PEP) binding sites is independent of pH in the range of 5.7-7.8. Variation of the Vmax value with pH indicates the presence of a dissociated group (pKa 6.2-6.3) and of an undissociated group (pKa 7.5-7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S0.5 value for this substrate, as is an undissociated enzyme group with a pKa in the range 6.7-7.0. It is shown that PEP and frucotse-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme, FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed.
Mol Biochem Parasitol 1981 Nov
PMID:Some kinetic properties of pyruvate kinase from Trypanosoma brucei: influence of pH and fructose-1,6-diphosphate. 732 90

Erythrocyte aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities are often used as indices of vitamin B-6 nutritional status; however, results using a mixed population of erythrocytes can be quite variable. Erythrocytes from two strains of mice (Mus domesticus), A/Ibg and DBA/Ibg, were separated according to age by centrifugation through discontinuous Percoll density gradients into three fractions: top (least dense, youngest), middle and bottom (most dense, oldest). A sufficient yield of age-fractionated erythrocytes was obtained from a single mouse for all of the enzyme measurements. The activities of AST, ALT and three age-marker enzymes, pyruvate kinase, acetylcholinesterase and hexokinase, were found to be significantly higher in the youngest cell fractions, and declined in the older, more dense fractions. A mice had significantly lower AST and ALT activities in the age separated fractions than did DBA mice. The measurement of enzyme activities in low density, young cells may be especially useful in studies involving conditions in which the proportion of young erythrocytes may be elevated with respect to the entire erythrocyte mass.
Comp Biochem Physiol B Biochem Mol Biol
PMID:Aminotransferase activities in mouse, Mus domesticus, erythrocytes separated according to age. 755 57

Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.
Mol Cell Biol 1995 Oct
PMID:Overproduction of a truncated hepatocyte nuclear factor 3 protein inhibits expression of liver-specific genes in hepatoma cells. 756 96

The activity and some kinetic parameters of the key enzymes of the glycolysis, the gluconeogenesis and the amino acid catabolism from the liver of male and female mink have been determined and compared to the corresponding activities from rat and cat. The activities of glucose-6-phosphatase and pyruvate kinase are dependent on sex, both being higher in females. Except for pyruvate carboxylase the glycolytic and the gluconeogenic enzyme activities of the mink are higher than those of rat and cat; especially the activities of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are markedly higher. The activities of glutamate dehydrogenase and glutamate oxaloacetate transaminase are smaller than the corresponding activities of rat but higher than those of cat. The results suggest that mink has a high capacity for gluconeogenesis compared to rat.
Comp Biochem Physiol B Biochem Mol Biol 1995 Sep
PMID:Activities of carbohydrate and amino acid metabolizing enzymes from liver of mink (Mustela vison) and preliminary observations on steady state kinetics of the enzymes. 758 47

Intravenous administration of a single dose (100 micrograms/kg bw) of recombinant tumour necrosis factor-alpha (TNF, cachectin) to rats increased the rate of in vitro fatty acid synthesis in interscapular brown adipose tissue (IBAT) from both glucose and alanine, without changes in the oxidation of these substrates to 14CO2. Lactate production and glycerol release were also unaffected by treatment with the cytokine. Additionally, the presence of TNF in the incubation media did not affect fatty acid synthesis, suggesting an indirect effect of the cytokine. The activities of different enzymes of glucose and alanine metabolism such as hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase and alanine transaminase, did not suffer changes as a consequence of TNF administration. The same applied to the enzymatic activities involved in fatty acid synthesis such as fatty acid synthase, acetyl-CoA carboxylase and ATP-citrate lyase. Conversely, citrate levels in IBAT were increased in animals treated with TNF, suggesting that it could be the cause for the increased fatty acid synthesis in this tissue.
Mol Cell Biochem 1995 Feb 23
PMID:Metabolic effects of tumour necrosis factor-alpha on rat brown adipose tissue. 759 46

We developed a new convenient method for determination of magnesium ion concentrations by use of magnesium ion-dependent enzymes; pyruvate kinase and lactate dehydrogenase. This method is based on the determination of the reaction rate of pyruvate kinase which uses MgADP- as substrate. The reaction rate of pyruvate kinase is dependent upon the formation of the complex with ADP and magnesium ion and the amount of the complex is dependent upon that of magnesium ion. The reaction rate of pyruvate kinase can be easily and spectrophotometrically determined by using lactate dehydrogenase and NADH as the decreased amount of absorbance at 340 nm.
Biochem Mol Biol Int 1995 Apr
PMID:Determination of magnesium ion by use of the coupled-enzyme method with pyruvate kinase and lactate dehydrogenase. 762 26

We have examined DNA from fifteen unrelated pyruvate kinase deficient patients with hereditary nonspherocytic hemolytic anemia (HNSHA) for the molecular alterations responsible for the enzyme deficiency. All but 3 of the 30 putative mutations were identified. Fourteen different mutations were found. Nine were missense mutations: 320 T-->C, 823 G-->C, 1276 C-->T, 1378 G-->A, 1484 C-->T, 1529 G-->A, 1654 G-->A, 1675 C-->G; three were nonsense mutations: 603 G-->A, 721 G-->T, 1501 C-->T; one was an insertion at 1574 GGG-->GGGG and the other a three nucleotide in-frame deletion 391-392-393 ATC. Eight of these mutations have not been previously described. We also investigated all of the patients for the C/A polymorphism at nt 1705 and the microsatellite ATT repeat in intron 11. All of the mutations that had previously been reported by us (391-393del, 721T, 1484T, 1529A) were found in the context of the same haplotype as the earlier cases, supporting the concept that each may have a single origin.
Blood Cells Mol Dis 1995
PMID:Study of the molecular defects in pyruvate kinase deficient patients affected by nonspherocytic hemolytic anemia. 765 61


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