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Query: EC:4.1.1.49 (phosphoenolpyruvate carboxykinase)
 4,654 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A previously described upstream hypersensitive site (HS) in the PEPCK gene at -4800 bp, termed HS A (1), has been characterized and determined to bind at least two factors. One of these is a member of the ubiquitous CREB/ATF family, and the second is a novel tissue specific protein, pep A. A construct carrying HS A and the PEPCK proximal promoter was tested in transgenic mice and its CAT activity compared to the proximal promoter alone. The HS A was shown to drive tissue-specific, position-independent transcription of the CAT reporter gene 2-3 fold more effectively than the proximal promoter alone, with a concommitant 4-5 fold higher expression of CAT. Protein binding activity has been localized to a 33 bp region. This region contains a CRE (2) which is shown to bind a member of the CREB/ATF family through competition assays with an oligo containing a CRE from the proximal promoter and by the appearance of a supershift when the factor/oligo complex was exposed to CREB polyclonal antibody. Through restriction enzyme digests and competition of protein binding with an oligonucleotide homologous to HS A with a mutated CRE we have characterized a putative binding site for a liver-specific factor. In vitro and 'in vivo' footprinting studies complement each other, as well as, mobility shift assay data in designating the binding site of the proteins. The CREB/ATF factor and Pep A bind independently of each other during short term incubations, however, both factors can be accomodated on the DNA substrate as a function of extended time of incubation. Preliminary biochemical analysis defines the subunit molecular mass of the CREB/ATF like proteins at 55, 42, and 35 kD, while the tissue specific material exists as a single homogeneous subunit polypeptide in SDS of molecular mass = 49 kD.
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PMID:Characterization of the factors binding to a PEPCK gene upstream hypersensitive site with LCR activity. 138 63

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.
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PMID:Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase. 277 22

A phosphoprotein of 65 kDa, as determined by SDS-gel electrophoresis, has been isolated from yeast crude extracts. This phospho form copurifies with phosphoenolpyruvate carboxykinase in the enzyme purification procedure worked out in our laboratory (Tortora, P., Hanozet, G.M. and Guerritore, A. (1985) Anal. Biochem. 144, 179-185). Moreover, both proteins bind strongly to 5'AMP-Sepharose 4B in the presence of Mn2+, whereas a substantially lower binding occurs if Mn2+ is replaced by Mg2+. This binding pattern is consistent with the well-known Mn2+-dependence of yeast phosphoenolpyruvate carboxykinase. These data suggest that the 65-kDa protein might be a phosphorylation product of the native enzyme. Furthermore, although the phospho form is not immunoprecipitated by anti-phosphoenolpyruvate carboxykinase antibodies, addition of Protein A-Sepharose CL-4B to crude extracts preincubated with the antibodies results in the binding to the resin of the phospho form, thus providing immunological evidence for its identification as a modified form of native enzyme. The same 65-kDa phosphoprotein is detectable in extracts from cells grown in the presence of [32P]Pi, as well as in cell extracts incubated with [gamma-32P]ATP. Moreover, digestion of the phosphoprotein with BrCN or with Staphylococcus aureus V8 proteinase, yields two and three fragments, respectively, which appear parallel to digestion products of phosphoenolpyruvate carboxykinase, again supporting the proposed identification. Finally, analysis of the phosphorylated amino acids in the 65-kDa protein shows that phosphoserine is the only labelled phosphoamino acid.
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PMID:Identification of a phosphorylated form of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae. 304 Jan 23

Day and night forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEPC) were extracted from leaves of the CAM plants Kalanchoe daigremontiana, K. tubiflora and K. blossfeldiana previously fed with [32P] labelled phosphate solution. A one-step immunochemical purification followed by SDS polyacrylamide gel electrophoresis and autoradiography showed that, in all species, the night form of the enzyme was phosphorylated and not the day form. Limited acid hydrolysis of the night form and two-dimensional separation identified predominantly labelled phosphoserine and phosphothreonine. In vitro addition of exogenous acid phosphatase (EC 3.1.3.2) to desalted night form-containing extracts resulted within 30 min in a shift in PEPC enzymic properties similar to the in vivo changes from night to day form. It is suggested that phosphorylation-dephosphorylation of the enzyme could be the primary in vivo process which might explain the observed rhythmicity of enzymic properties.
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PMID:Phosphorylation-dephosphorylation process as a probable mechanism for the diurnal regulatory changes of phosphoenolpyruvate carboxylase in CAM plants. 370 71

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.
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PMID:Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana. 397 Sep 41

Phosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70,000 by SDS-gel electrophoresis, 65,000 by Sephadex G-100 gel filtration and 72,000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 microM for GTP, 8.5 microM for GDP, and 171 microM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.
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PMID:Purification and molecular characteristics of mitochondrial phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver. 697 Jan 95

Cyclic-AMP stabilizes phosphoenolpyruvate carboxykinase (GTP) (PEPCK) mRNA against degradation. To investigate the mechanism of this effect, RNA mobility shift assays were used to determine the interaction of cellular proteins with specific domains from the mRNA. We report here the identification of a protein with an affinity for sequences of PEPCK mRNA with a predicted stem-loop structure. RNA-protein complex formation was significantly reduced if the double-stranded RNA probe was preheated to 90 degrees C. The RNA-binding protein did not bind to the hairpin structure of poly(rI)-poly (rC), indicating some degree of sequence specificity and that the RNA-binding protein is not the interferon-induced double-stranded RNA-activated protein kinase. The binding activity was contained in the cytosolic fraction (100,000 x g) of rat hepatoma FTO-2B cells and was significantly enhanced by high concentrations of KCl. Chromatography on an anion exchanger separated the binding activity from a factor which, upon reconstitution, inhibited the interaction with the RNA probe. Incubation of cells with cAMP resulted in a 3-4-fold decrease in the activity of the RNA-binding protein. An inhibition in complex formation was observed with extracts as early as 60 min after exposure of cells to cAMP. Liver extracts from rats starved for 72 h also had reduced binding activity compared to extracts from fed animals. Cellular extracts treated with alkaline phosphatase exhibited an elevated level of complex formation. An analysis by SDS-polyacrylamide gel electrophoresis of the RNA-protein complex after ultraviolet light cross-linking demonstrated that the RNA-binding protein had a molecular mass of approximately 100 kDa. On the basis of these results, we suggest that liver cells contain a protein whose interaction with PEPCK mRNA is regulated by cAMP-dependent phosphorylation and which may be responsible for the cAMP-mediated control of PEPCK mRNA half-life.
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PMID:A cAMP-regulated RNA-binding protein that interacts with phosphoenolpyruvate carboxykinase (GTP) mRNA. 822 67

Clostridium symbiosum pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The nucleotide binding site of this enzyme was labeled using the photoaffinity reagent [32P]-8-azidoadenosine 5'-triphosphate ([32P]-8-azidoATP). Subtilisin cleavage of the [alpha-32P]-8-azidoATP-photolabeled PPDK into domain-sized fragments, prior to SDS-PAGE analysis, allowed us to identify two sites of modification: one between residues 1 and 226 and the other between residues 227 and 334. Saturation of the ATP binding site with adenylyl imidodiphosphate afforded protection against photolabeling. Next, small peptide fragments of [gamma-32P]- 8-azidoATP-photolabeled PPDK were generated by treating the denatured protein with trypsin or alpha-chymotrypsin. A pair of overlapping radiolabeled peptide fragments were separated from the two digests, DMQDMEFTIEEGK (positions 318-330 in trypsin-treated PPDK) and RDMQDMEFTIEEGKL (positions 317-331 in alpha-chymotrypsin-treated PPDK), thus locating one of the positions of covalent modification. Next, catalysis by site-directed mutants generated by amino acid replacement of invariant residues of the PPDK N-terminal domain was tested. K163L, D168A, D170A, D175A, K177L, and G248I PPDK mutants retained substantial catalytic activity while G254I, R337L, and E323L PPDK mutants were inhibited. Comparison of the steady-state kinetic constants measured (at pH 6.8, 25 degrees C) for wild-type PPDK (kcat = 36 s-1, AMPK(m) = 7 microM, PP(i)K(m) = 70 microM, PEPK(m) = 27 microM) to those of R337L PPDK (kcat = 2 s-1, AMPK(m) = 85 microM, PP(i)K(m) = 3700 microM, PEPK(m) = 6 microM) and G254I PPDK (kcat = 0.1 s-1, AMPK(m) = 1300 microM, PP(i)K(m) = 1200 microM, PEPK(m) = 12 microM) indicated impaired catalysis of the nucleotide partial reaction (E.ATP.P(i) --> E-PP.AMP.P(i) --> E-P.AMP.PP(i) in these mutants. The single turnover reactions of [32P]PEP to [32P]E-P.pyruvate catalyzed by the PPDK mutants were shown to be comparable to those of wild-type PPDK. In contrast, the formation of [32P]E-PP/[32P]E-P in single turnover reactions of [beta-32P]ATP/P(i) was significantly inhibited. Finally, the location of the adenosine 5'-diphosphate binding site within the nucleotide binding domain of D-alanine-D-alanine ligase, a structural homologue of the PPDK N-terminal domain [Herzberg, O. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 2652-2657] indicates, by analogy, the location of the nucleotide binding site in PPDK. Residues G254, R337, and E323 as well as the site of photoaffinity labeling are located within this region.
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PMID:Determination of the nucleotide binding site within Clostridium symbiosum pyruvate phosphate dikinase by photoaffinity labeling, site-directed mutagenesis, and structural analysis. 867 15

We have previously shown that phosphoenolpyruvate carboxykinase (PEPCK) is phosphorylated in vivo in the cotyledons of darkened cucumber seedlings and that phosphorylation is reversed by light [Walker and Leegood (1995) FEBS Lett. 362, 70-74]. In this study the molecular mass of PEPCK was estimated in a range of gluconeogenic seedlings and in leaves of C4 plants and plants with Crassulacean acid metabolism (CAM). Phosphorylation of PEPCK was studied in these plants by feeding tissues with [32P]Pi and assessing phosphorylation by SDS/PAGE and autoradiography of either total proteins or of immunoprecipitated protein. In gluconeogenic seedlings and most CAM plants PEPCK had a molecular mass of 74 kDa, whereas in C4 grasses the molecular mass of PEPCK was always smaller and varied from 67-71 kDa. In all gluconeogenic seedlings and leaves of CAM plants PEPCK was phosphorylated, but it was not phosphorylated in all species of C4 grasses studied. In CAM plants, phosphorylation of PEPCK occurred at night and dephosphorylation occurred during the day. In C4 grasses phosphorylation occurred when leaves were darkened and the enzyme was dephosphorylated following illumination, but it was only phosphorylated in those plants with larger (71 kDa) molecular mass forms of PEPCK.
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PMID:Phosphorylation of phosphoenolpyruvate carboxykinase in plants. Studies in plants with C4 photosynthesis and Crassulacean acid metabolism and in germinating seeds. 876 Mar 46

Four isoforms of phosphoenolpyruvate carboxylase (PEPC1, PEPC2, PEPC3, PEPC4) have been purified from the green alga Selenastrum minutum. PEPC1 is a homotetramer with a subunit M(r) of 102 kDa. PEPC2, PEPC3, and PEPC4 have respective native M(r)S of approximately 984, 1186, and 1590 kDa. SDS/PAGE analysis revealed that the latter three isoforms contain polypeptides having M(r)S of 102, 73, 70, 65, and 61 kDa. Immunoblot analyses and CNBr cleavage patterns suggest that the 102-kDa polypeptide present in all four isoforms is the same PEPC catalytic subunit. Our data suggest that the three high M(r)S PEPC isoforms are heteromeric protein complexes consisting of the 102-kDa PEPC1 catalytic subunit and immunologically unrelated polypeptides. Attempts to measure other enzyme activities associated with the protein complexes gave negative results. However, PEPC1 had immunological, physical, and kinetic properties very different from those of the larger M(r) PEPC isoforms: (i) the anti-PEPC1 immune-serum was relatively inefficient for immunoprecipitating PEPC2, PEPC3, or PEPC4; (ii) immune-serum raised against a mixture of PEPC2, PEPC3, and PEPC4 had relatively weak immunoprecipitating activity toward PEPC1; (iii) PEPC1 was more heat sensitive than the other three isoforms; (iv) PEPC1 had a pH optimum of 9 versus 8.5 for the PEPC protein complexes; (v) the high Mr PEPCs had greater apparent affinity for phosphoenolpyruvate compared to PEPC1 and (vi) PEPC1 activity was far more sensitive to metabolite activators (Gln and dihydroxyacetone phosphate) and inhibitors (Asp, Glu, 2-oxoglutarate and malate). We conclude that the interaction of the PEPC catalytic subunit with unrelated polypeptides is responsible for the observed differences between PEPC1 and the high molecular mass isoforms. We propose that this interaction possibly regulates PEPC activity in vivo.
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PMID:Purification and properties of four phosphoenolpyruvate carboxylase isoforms from the green alga Selenastrum minutum: evidence that association of the 102-kDa catalytic subunit with unrelated polypeptides may modify the physical and kinetic properties of the enzyme. 880 8


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