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Query: EC:4.1.1.32 (phosphoenolpyruvate carboxykinase)
 4,204 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphoenolpyruvate carboxykinase activity in crude extracts of muscle has frequently been determined by using a continuous spectrophotometric method, which is shown to grossly overestimate enzyme activity. NADH oxidation attributed to phosphoenolpyruvate carboxykinase activity in the assay is due to lactate production. Under the normal assay conditions. Na+ ions stimulate pyruvate kinase, providing pyruvate for lactate formation by lactate dehydrogenase and sufficiently to account for most of the observed NADH oxidation.
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PMID:Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle. 628 11

The effect of N6,O2'-dibutyryl cyclic adenosine monophosphate (Bt2cAMP) on the induction of the mRNA coding for the enzyme phosphoenolpyruvate carboxykinase was examined in H4-II-E cells. this mRNA comprised about 0.1% of total cellular poly(A)+RNA activity in uninduced cells and was increased 5- to 7-fold by the cyclic nucleotide. The maximal level was reached 3 h after addition of the nucleotide to the cell culture. This induction is attributed to cAMP since the nonmetabolizable analogs 8-bromocAMP and 8-(4-chlorophenylthio)cAMP produce inductions comparable to Bt2cAMP while sodium butyrate and dibutyryl cyclic GMP had little effect. The increased translational activity correlated well with a proportionate increase in the amount of phosphoenolpyruvate carboxykinase (P-enolpyruvate carboxykinase) mRNA sequences which were hybridizable to a specific cDNA probe. Blot hybridization of total nuclear RNA isolated from uninduced H4-II-E cells revealed eight P-enolpyruvate carboxykinase RNA sequence species ranging in size from 1.8 to 6.9 kilobases. Treatment with Bt2cAMP increased the amount of all eight of these forms. This increase became maximal by 45-60 min and was maintained for at least 1 h. In contrast, analysis of cytoplasmic RNA showed a single 3.2-kilobase (23 S) band, which was still increasing in amount 2 h after Bt2cAMP treatment. Thus, Bt2cAMP resulted in a sequential induction of nuclear P-enolpyruvate carboxykinase RNA sequences followed by an increase in cytoplasmic phosphoenolpyruvate carboxykinase mRNA. We conclude that cyclic AMP exerts its main effect on P-enolpyruvate carboxykinase induction at the nuclear level.
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PMID:Induction of the messenger ribonucleic acid coding for phosphoenolpyruvate carboxykinase in H4-II-E cells. Evidence for a nuclear effect of cyclic AMP. 629 24

Insulin causes a 7-10-fold decrease of both the mRNA that codes for rat hepatic phosphoenolpyruvate carboxykinase (mRNAPEPCK) and of PEPCK synthesis, provided the animals are made diabetic and fed chow. mRNAPEPCK, measured either by in vitro translation or cDNA hybridization, decreases with a half-time of 30-60 min after insulin treatment. This coordinant decrease, which approximates the half-life of mRNAPEPCK measured in a variety of situations, suggests that insulin acts by decreasing mRNAPEPCK production, and that the hormone does not alter the activity of a fixed amount of this RNA, or enhance its degradation. Glucagon results in a ninefold induction of mRNAPEPCK. Half-maximal induction occurs with doses between 20-75 micrograms/100 g body wt and occurs within 30-45 min. Maximal induction requires 150 micrograms/100 g body wt and occurs about 80 min after a single glucagon injection. N6,O2'-dibutyryl cAMP and a cAMP analogue that is not metabolized, 8-(4-chlorophenyl-thio)cAMP, induce mRNAPEPCK as effectively as glucagon and with similar kinetics. Since sodium butyrate, adenosine, and dibutyryl cGMP are ineffective inducers, cAMP appears to be the active agent in the hepatocyte.
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PMID:Insulin and glucagon regulate cytosolic phosphoenolpyruvate carboxykinase (GTP) mRNA in rat liver. 632 36

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.
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PMID:Phosphoenolpyruvate carboxykinase from guinea-pig liver mitochondria. Immunological evidence for increase in enzyme amount during neonatal development. 643 67

Highly purified glycosomes were isolated from Trypanosoma brucei bloodstream forms and cultured procyclic trypomastigotes. A comparison of the specific activities of glycosomal enzymes revealed that glycosomes from insect stages had decreased levels of hexokinase, phosphoglucose isomerase, phospho-fructokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase, but contained increased levels of adenylate kinase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. Glycosomes from bloodstream forms were almost totally devoid of the latter two activities. Comparison of the two types of glycosomes by sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that bloodstream form glycosomes contained 3 prominent polypeptides (64, 46 and 40 kDa) which were hardly detectable in insect stage glycosomes, whereas the latter contained 3 insect stage specific bands with molecular weight of 34 000, 61 000 and 77 000 and 4 additional bands with molecular weights between 94 000 and 110 000. Both types of glycosome contained the phospholipids phosphatidylcholine and phosphatidylethanolamine. Insect stage glycosomes contained in addition also phosphatidylinositol and some phosphatidylserine.
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PMID:A comparison of the glycosomes (microbodies) isolated from Trypanosoma brucei bloodstream form and cultured procyclic trypomastigotes. 674 87

The adsorption of Escherichia coli phosphoenolpyruvate carboxylase [EC 4.1.1.31] to butyl-, hexyl-, and octyl-Sepharose gels was investigated. The enzyme was nearly completely adsorbed to the latter two gels both in the absence and presence of high concentrations of ammonium sulfate. At intermediate concentrations--0.1 M in the case of hexyl-Sepharose--virtually no adsorption was observed. Upon application of an increasing or decreasing concentration gradient of the salt, the enzyme was eluted at various concentrations of the salt depending on chain length of the immobilized alkyl groups. The adsorption to hexyl-Sepharose at 0.7 M ammonium sulfate was markedly decreased by L-aspartate, the allosteric inhibitor, whereas it was increased by acetyl-CoA, one of the allosteric activators. Evidence was obtained suggesting that these changes in adsorption were due to conformational alterations of the enzyme elicited by these effectors. The enzyme seemed to have been adsorbed at its hydrophobic regions which were distinct from the allosteric site for long-chain fatty acids. The specific elution with L-aspartate in the presence of 0.82 M ammonium sulfate could successfully be applied to purification of the enzyme. By this hydrophobic interaction chromatography, the enzyme was purified about 55-fold over its partially purified preparation with a recovery of 73%. The obtained enzyme preparation was almost homogeneous as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis.
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PMID:Phosphoenolpyruvate carboxylase of Escherichia coli. Hydrophobic chromatography using specific elution with allosteric inhibitor. 675 31

Incubation of partially purified yeast phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) with 5% mercaptoethanol and 0.01% sodium dodecyl sulfate at 37 degrees C results in degradation of the enzyme. The degradation can be partially prevented by addition of proteinase B inhibitor 2 or phenylmethylsulfonyl fluoride, an inhibitor of proteinase B and carboxypeptidase Y. The degradation can be completely inhibited by addition of proteinase B inhibitor 2 together with pepstatin, and inhibitor of proteinase A. Thus it appears that proteolytic activities are firmly attached to phosphoenolpyruvate carboxykinase and are identical with the yeast proteinases A and B. The latter conclusion was supported by experiments using the pure yeast proteinases.
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PMID:Characterization of the proteolytic activity firmly attached to yeast phoshoenolpyruvate carboxykinase. 705 35

The enzyme phosphoenolpyruvate carboxykinase has been purified from chicken liver mitochondria. This purification includes a pseudo-affinity column step utilizing Sepharose 4B-blue dextran which binds the enzyme. The enzyme elutes with ITP to yield protein which is greater than 98% pure. The enzyme has Mr = 75,400 +/- 200 estimated by high speed sedimentation equilibrium and 70,500 +/- 500 estimated by reduced sodium dodecyl sulfate-polyacrylamide gels. The enzyme is abnormally retarded on molecular exclusion resins yielding low apparent molecular weight values. The amino acid analysis indicates that the enzyme has a high proline content and a high tryptophan content and contains 9 mol of cysteine/mol of enzyme. No disulfide bonds were detected. The extinction coefficient (epsilon 1% 280 = 16.5 +/- 0.1) reflects the high tryptophan content. The Svedberg coefficient (s20,w = 4.63 +/- 0.03 S) is consistent with a globular protein of Mr = 70,500-75,400. The activation of the enzyme was investigated by steady state kinetics. The carboxylation reaction has an activation energy of 17.6 kcal/mol. There is no requirement of a monovalent cation for activity. A thiol is necessary for maximal activity, although apparently not to reduce disulfide bonds within the enzyme. Incubation with dithiothreitol stabilizes enzymatic activity but beta-mercaptoethanol facilitates loss of activity. The kinetics of activation by Mn2+ was performed. The Ks value for phosphoenolpyruvate (300 microM) decreases to an apparent Km of 67 microM with increasing concentrations of Mn2+. The concentration of Mn2+ does not affect the interaction of HCO-3 with the enzyme, however. Analysis of data in terms of free IDP indicates that increasing Mn2+ decreases the Km of IDP but analysis as MnIDP indicates the Km,app of MnIDP is independent of the Mn2+ concentration. The enzyme interacts with Mn2+ with a KA = 67 microM and the Km,app decreases to a value of 8 microM with saturating substrates. The substrate analogue (Z)-3-fluorophosphoenolpyruvate is a good substrate for the reaction (Km = 30 microM) with 27% Vmax compared to P-enolpyruvate (Km = 180 microM). Except for 3-bromophosphoenolpyruvate, other analogues have shown weak competitive or noncompetitive inhibition. Potential analogues of oxalacetate (succinate, citrate, isocitrate, malate, and alpha-ketoglutarate) all elicit weak (greater than 15 mM) inhibition.
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PMID:The purification, characterization, and activation of phosphoenolpyruvate carboxykinase from chicken liver mitochondria. 706 3

The relationship among sodium transport (TNa), oxygen consumption (QO2), and gluconeogenesis was studied in isolated perfused rat kidneys in which glucose formation was enhanced by providing pyruvate as a substrate and by prior treatment with methylprednisolone. TNa was increased abruptly by increasing perfusion pressure as to increase GFR or by lowering the albumin concentration of a hyperoncotic perfusate as to allow glomerular filtration to occur. Increases in TNa of 40% were accompanied by little or no increase in QO2, whereas gluconeogenesis decreased 55-80%. Conversely, a decrease in perfusion pressure that lowered TNa produced an increase in glucose formation without a change in QO2. When gluconeogenesis was blocked with 0.15 mM 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, QO2 increased together with TNa as perfusion pressure was raised. The results suggest that the energy needed for ion transport by the kidney may under some circumstances be borrowed from nontransport functions and, therefore, that basal oxygen consumption may vary with the rate of reabsorptive transport.
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PMID:Relationship among gluconeogenesis, QO2, and Na+ transport in the perfused rat kidney. 708 37

Polyadenylated RNA has been isolated from maize leaves at various times during greening of etiolated seedlings and used to prime the wheat germ cell-free translation system. Levels of translatable messenger RNA for phosphoenolpyruvate carboxylase increase with the length of the illumination period. The pattern of the increase in translatable mRNA for the enzyme is similar to that of the increase in phosphoenolpyruvate carboxylase protein previously observed in intact tissues. Phosphoenolpyruvate carboxylase synthesized in vivo migrates as a doublet band on gradient sodium dodecyl sulfate-polyacrylamide gels. A similar doublet has been seen occasionally with the products of cell-free translation.
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PMID:Light-stimulated increase of translatable mRNA for phosphoenolpyruvate carboxylase in leaves of maize. 726 49


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