Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

The cytosolic form of phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) from rat liver was purified by a procedure involving affinity chromatography on agarose-hydrazide-GTP. Phosphoenolpyruvate carboxykinase is retained quantitatively by the affinity medium in the presence of manganese and can be specifically eluted by a pulse of GTP. On the contrary, no binding to agarose-hydrazide-GTP occurs in the absence of manganese. This suggests that the affinity of the enzyme for GTP is enhanced by prior interaction with manganese. A combination of several conventional purification steps followed by affinity chromatography provides pure phosphoenolpyruvate carboxykinase in good yields. The final specific activity is 19 U/mg protein. The enzyme migrates as a single polypeptide of molecular weight 70,600 during electrophoresis on sodium dodecyl sulfate polyacrylamide gels.
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PMID:Purification of phosphoenolpyruvate carboxykinase (GTP) by affinity chromatography on agarose-hydrazide-GTP. 52 Feb 80

We have cloned and sequenced the pckA gene of Rhizobium sp. NGR234, a broad host-range strain. The gene encodes phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of gluconeogenesis. The locus was isolated and subcloned from a genomic library of NGR234 employing hybridization with an R. meliloti pck gene probe and complementation of a Tn5 mutant in this species. The DNA sequence of pckA (NGR234) was determined and encoded a PEPCK protein of 535 amino acids with a molecular weight of 58.4 kDa. The deduced polypeptide sequence was compared to those of three known ATP-dependent PEPCKs. Slightly higher homology was observed with yeast and trypanosome polypeptides than with that of Escherichia coli. We have identified several regions that are conserved in all four PEPCK proteins. A mutant constructed in the pck gene by site-directed mutagenesis with interposon omega failed to grow on succinate, malate and arabinose but grew on glucose and glycerol as sole carbon sources. These data show that NGR234 requires PEPCK-driven gluconeogenesis to grow on TCA cycle intermediates. A host-dependent effect of the pckA mutation was observed on nodule development and nitrogen fixation. Nodules formed by the site-directed mutant on Leucaena leucocephala and Macroptilium atropurpureum were FixRed, but on Vigna unguiculata were Fix-. The expression of the gene was positively regulated in free-living cells of NGR234 by either succinate or host-plant exudates, and was subject to catabolite repression by glucose.
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PMID:Site-directed mutagenesis and DNA sequence of pckA of Rhizobium NGR234, encoding phosphoenolpyruvate carboxykinase: gluconeogenesis and host-dependent symbiotic phenotype. 172 Aug 62

Extracts of the leaf tissue of Panicum maximum Jacq. var. trichoglume Eyles (a phosphoenolpyruvate carboxykinase type of C4 plant) were examined and at least two isoforms of aspartate aminotransferase (EC 2.6.1.1), with different electrophoretic mobilities, were detected. The predominant isoform was purified to homogeneity from mesophyll cells. The purification procedure included fractionation with ammonium sulfate followed by chromatography on diethylaminoethyl-cellulose, Sephacryl S-300, and hydroxyapatite. The purified enzyme had specific activities of 182 and 165 mumol/min/mg protein, measured in terms of the synthesis of oxaloacetate and aspartate, respectively, at pH 8.0. The enzyme, with an apparent molecular size of 100 kDa, appears to be a dimer of a single polypeptide with a molecular size of 42 kDa. Mono specific polyclonal antibodies were raised against the 42-kDa polypeptide. Only a single stained band was detected in extracts of whole leaves by immunoblot analysis with this antibody after two-dimensional polyacrylamide electrophoresis. Furthermore, no difference in mobility was observed between the enzymes extracted from mesophyll and bundle sheath cells on native polyacrylamide gels. These findings are discussed in relation to the other isoform in the leaves of this species.
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PMID:Aspartate aminotransferase from Panicum maximum Jacq. var. trichoglume Eyles, a C4 plant: purification, molecular properties, and preparation of antibody. 293 Jan 93

Insulin is an anabolic polypeptide hormone with pleiotrophic effects. During the decades since the initial description by Banting and Best, the acute effects of insulin have been widely studied with particular focus on the mechanism or mechanisms of insulin activation of hexose transport and regulation of metabolic enzyme activity. However, recently there has been a major expansion of investigation to include insulin regulation of gene expression with multiple insulin-sensitive specific mRNAs now reported. In this review, we explore the involvement of insulin-induced changes in plasma membrane glycerolipid metabolism in the transmembrane signaling process required for insulin regulation of mRNA levels. Insulin increases diacylglycerol levels in insulin-responsive cells, and synthetic diacylglycerols or their phorbol ester diacylglycerol analogs, such as 4 beta,9 alpha,12 beta,13 alpha, 20-pentahydroxytiglia-1,6-dien-3-one 12 beta-myristate 13-acetate (TPA), mimic insulin regulation of ornithine decarboxylase mRNA, c-fos mRNA, and phosphoenolpyruvate carboxykinase mRNA levels. This suggests that insulin regulation of specific mRNA levels may be mediated by insulin-induced changes in phospholipid metabolism and that diacylglycerol may play a pivotal role in insulin regulation of gene expression.
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PMID:Insulin-glycerolipid mediators and gene expression. 328 49

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.
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PMID:The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence. 608 98

Mice homozygous for the lethal spotting (ls) mutation exhibit aganglionic megacolon and a white spotted coat owing to a lack of neural crest-derived enteric ganglia and melanocytes. The ls mutation disrupts the migration, differentiation, or survival of these neural crest lineages during mammalian development. A human congenital disorder, Hirschsprung disease (HSCR), is also characterized by aganglionic megacolon of the distal bowel and can be accompanied by hypopigmentation of the skin. HSCR has been attributed to multiple loci acting independently or in combination. The ls mouse serves as one animal model for HSCR, and the ls gene may represent one of the loci responsible for some cases of HSCR in humans. This study uses 753 N2 progeny from a combination of three intersubspecific backcrosses to define the molecular genetic linkage map of the ls region and to provide resources necessary for positional cloning. Similar to some cases of HSCR, the ls mutation acts semidominantly, its phenotypic effects dependent upon the presence of modifier genes segregating in the crosses. We have now localized the ls mutation to a 0.8-cM region between the D2Mit113 and D2Mit73/D2Mit174 loci. Three genes, endothelin-3 (Edn3), guanine nucleotide-binding protein alpha-stimulating polypeptide 1 (Gnas), and phosphoenolpyruvate carboxykinase (Pck1) were assessed as candidates for the ls mutation. Only Edn3 and Gnas did not recombine with the ls mutation. Mutational analysis of the Edn3 and Gnas genes will determine whether either gene is responsible for the neural crest deficiencies observed in ls/ls mice.
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PMID:A high-resolution linkage map of the lethal spotting locus: a mouse model for Hirschsprung disease. 771 19

A rabbit antiserum was raised against phosphoenolpyruvate carboxykinase (PCK) purified from Urochloa panicoides, a PCK-type C4 monocot. The antiserum was used to screen a cDNA expression library constructed from U. panicoides leaf poly(A)+RNA. Inserts from immunoreactive clones were used to rescreen the library and obtain three overlapping cDNAs comprising a 2220 bp composite sequence. The single complete open reading frame of 1872 bp encodes PCK1, a 624 amino acid polypeptide with a predicted molecular mass of 68,474 Da. Comparison of PCK1 with other ATP-dependent PCKs indicates that PCK1 is significantly larger, mainly due to an N-terminal extension of greater than 65 residues, and reveals high sequence identity across the central portion of the protein, especially over seven sub-sequences. One of these sub-sequences spans motifs common to several ATP-utilising enzymes for phosphate and divalent cation binding. The anti-PCK antiserum recognises a 69 kDa polypeptide on immunoblots of either purified PCK or U. panicoides leaf extracts. However, polypeptides of 63, 62, 61 and 60 kDa are also immunoreactive. Amino terminal sequencing of polypeptides from preparations of purified PCK demonstrates that these smaller polypeptides are related to PCK1, and time course experiments show that these polypeptides arise from the breakdown of PCK during isolation. Northern blot analysis indicates that the 2.7 kb PCK mRNA is abundant in green leaves but not in roots or etiolated shoots. Moreover, PCK mRNA levels increase gradually during greening, reaching maximum levels after about 84 h.
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PMID:Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides. 788 25

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.
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PMID:Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression. 794 88

The complete structural gene for argininosuccinate lyase (argH) from Campylobacter jejuni TGH9011 has been cloned into Escherichia coli by complementation of an E. coli argH auxotrophic mutant. The gene has been subcloned for sequencing on a 4.1-kb DNA segment and localized by the complementing activity of deletion mutants. The complete DNA sequence of the C. jejuni argH gene was determined. The transcription start point for argH mRNA was determined by primer extension analysis and found to be within the coding sequence of the upstream gene, identified as the phosphoenolpyruvate carboxykinase gene (ppc). The argininosuccinate lyase and the phosphoenolpyruvate carboxykinase reading frames overlap by one base, the second example of this phenomenon in C. jejuni chromosomal genes. The enzyme has a deduced subunit molecular weight of 51,831. Recombinant plasmids containing the argH gene generate a 56-kDa protein and a 43-kDa protein in E. coli maxicells. An alternate translation initiation producing a polypeptide with a deduced molecular mass of 42 kDa may account for the smaller protein observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C. jejuni argH gene shows nucleotide homology to both yeast and human argininosuccinate lyase genes, and conserved amino acid domains are evident between the corresponding proteins.
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PMID:Cloning, characterization, and nucleotide sequence analysis of the argH gene from Campylobacter jejuni TGH9011 encoding argininosuccinate lyase. 814 52

Following in situ renaturation and assay of protein kinase activity after denaturing electrophoresis of relatively impure samples of maize phosphoenolpyruvate carboxylase (PEPC) kinase, a approximately 30-kDa polypeptide was implicated as the best candidate for the PEPC kinase catalytic subunit. This kinase's apparent native molecular weight was estimated at 28,000 by gel filtration on a calibrated Superose 12 column (HR 10/30), suggesting that the isolated PEPC kinase is monomeric. This protein-serine kinase was partially purified about 4000-fold from illuminated maize leaves by ammonium sulfate precipitation and sequential chromatography on Ultrogel AcA 54, hydroxylapatite, blue dextran-agarose, and an analytical AcA 54 column. Analysis by denaturing electrophoresis revealed that a 30-kDa polypeptide copurified with PEPC kinase activity during the final step. This highly purified kinase had an apparent Km (PEPC subunit) of 2.5 microM and a Km (total ATP) of 40 microM at pH 8.0, its pH optimum. Upon in vitro phosphorylation of darkform (dephospho) C4 PEPC at Ser-15 (maize PEPC) or Ser-8 (sorghum), the malate sensitivity of the target enzyme decreased significantly. The maize PEPC kinase activity was markedly inhibited by L-malate, a negative allosteric effector of its protein substrate, in a concentration- and pH-dependent manner. Comparative phosphorylation studies with the catalytic subunit of mammalian cAMP-dependent protein kinase and casein revealed that a significant part of the malate inhibition of PEPC kinase activity in vitro was due to this effector's interaction with PEPC. The activity of both the highly purified PEPC kinase and a less pure sample prepared rapidly in the presence of various protease inhibitors was insensitive to Ca2+ chelation or addition. It is concluded that the approximately 30-kDa maize PEPC kinase is a low abundance, Ca(2+)-independent protein-serine kinase that activates its target enzyme by the exclusive phosphorylation of the regulatory serine residue near the N terminus and the resulting decrease in feedback inhibition by L-malate.
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PMID:Partial purification and characterization of phosphoenolpyruvate carboxylase protein-serine kinase from illuminated maize leaves. 834 24


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