Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P67775 (alpha isoform)
 797 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.
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PMID:Purification and characterization of a novel xylulose 5-phosphate-activated protein phosphatase catalyzing dephosphorylation of fructose-6-phosphate,2-kinase:fructose-2,6-bisphosphatase. 759 45

Okadaic acid (2 nM) inhibited by 80-90% the protein phosphatase activities in diluted extracts of rat liver, human fibroblasts, and Xenopus eggs acting on three substrates (high mobility group protein-I(Y), caldesmon and histone H1) phosphorylated by a cyclin-dependent protein kinase (CDK) suggesting that a type-2A phosphatase was responsible for dephosphorylating each protein. This result was confirmed by anion exchange chromatography of rat liver and Xenopus extracts, which demonstrated that the phosphatases acting on these substrates coeluted with the two major species of protein phosphatase 2A, termed PP2A1 and PP2A2. When matched for activity toward glycogen phosphorylase, PP2A1 was five- to sevenfold more active than PP2A2 and 35-fold to 70-fold more active than the free catalytic subunit (PP2Ac) toward the three CDK-labeled substrates. Protein phosphatases 1, 2B, and 2C accounted for a negligible proportion of the activity toward each substrate under the assay conditions examined. The results suggest that PP2A1 is the phosphatase that dephosphorylates a number of CDK substrates in vivo and indicate that the A and B subunits that are associated with PP2Ac in PP2A1 accelerate the dephosphorylation of CDK substrates, while suppressing the dephosphorylation of most other proteins. The possibility that PP2A1 activity is regulated during the cell cycle is discussed.
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PMID:Protein phosphatase 2A1 is the major enzyme in vertebrate cell extracts that dephosphorylates several physiological substrates for cyclin-dependent protein kinases. 840 Apr 54

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.
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PMID:Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa. 852 28

Methylotrophic yeast Pichia pastoris was used for a medium-scale expression of structural (PR65/A) and catalytic (PP2Ac) subunits of human type 2A protein phosphatase (PP2A). Constructs encoding these subunits, which were designed to introduce eight histidines at their N-termini, were introduced into the KM71 Pichia strain by homologous recombination. Recombinant proteins overproduced after methanol induction were purified from cell-free extracts by anion-exchange chromatography on DEAE-Sepharose, and Ni2+/nitrilotriacetate/agarose. In addition, chromatography on omega-aminohexyl-Sepharose was applied to purify recombinant (r)PR65/A. This purification scheme yielded approximately 5 mg and 100 microg of rPR65/A and rPP2Ac, respectively, from 1 L of the yeast culture. The specific activity of rPP2Ac measured with [32P]phosphorylase a [1.7 micromol.min-1.(mg protein)-1] and its inhibition by okadaic acid (IC50 = 0.66 nM) were similar to PP2A isolated from rabbit skeletal muscle. As demonstrated by immunodetection with methylation state-specific antibodies, recombinant PP2Ac was carboxymethylated at the last C-terminal leucine residue. Recombinant PP2A subunits were able to form a complex as demonstrated both by activity assays in the presence of protamine and by chromatography on protamine-agarose. In summary, P. pastoris provides a convenient heterologous system for the production of recombinant subunits of PP2A.
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PMID:Biochemical characterization of recombinant subunits of type 2A protein phosphatase overexpressed in Pichia pastoris. 1093 Dec 6

The activity and allosteric properties of plant phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved serine residue near the N-terminus. This up/down-regulation of PEPC is catalyzed by a dedicated and highly regulated serine/threonine (Ser/Thr) kinase (PEPC-kinase) and an opposing type-2A Ser/Thr phosphatase (PP2A). In marked contrast to PEPC-kinase, the PP2A holoenzyme from photosynthetic tissue has been virtually unstudied to date. In the present investigation, we have partially purified and characterized the native form of this PP2A from illuminated leaves of maize (Zea mays L.), a C4 plant, using maize [32P]PEPC as substrate. Various conventional chromatographic matrices, together with thiophosphorylated C4 PEPC-peptide and microcystin-LR affinity-supports, were exploited for the enrichment of this PP2A from soluble leaf extracts. Biochemical and immunological results indicate that the C4-leaf holoenzyme is analogous to other eukaryotic PP2As in being a approximately 170-kDa heteromer comprised of a core PP2Ac-A heterodimer (approximately 38- and approximately 65-kDa subunits, respectively) complexed with a putative, approximately 74-kDa B-type regulatory/targeting subunit. This heterotrimer lacks any strict substrate specificity in that it dephosphorylates C4 PEPC, mammalian phosphorylase a, and casein in vitro. This activity is independent of free Me2+, insensitive to levamisole and the Inhibitor-2 protein that targets PP1, activated by several polycations such as protamine and poly-L-lysine, and highly sensitive to inhibition by microcystin-LR and okadaic acid (IC50 approximately 30 pM), all of which are diagnostic features of yeast and mammalian PP2As. In addition, this C4-leaf PP2A holoenzyme (i) is inhibited in vitro by physiological concentrations of certain C4 PEPC-related metabolites (L-malate, PEP, glucose 6-phosphate, but not the activator glycine) when either 32P-labeled maize PEPC or rabbit muscle phosphorylase a is used as substrate, suggesting a direct effect on this Ser/Thr phosphatase; and (ii) displays, at best, only modest light/dark effects in vivo on its apparent molecular mass, component core subunits and activity against C4 PEPC, in marked contrast to the opposing activity of PEPC-kinase in C4 and Crassulacean acid metabolism leaves. This report represents one of the few studies of a heteromeric PP2A holoenzyme from photosynthetic tissue that dephosphorylates a known target enzyme in plants, such as PEPC, sucrose-phosphate synthase or nitrate reductase.
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PMID:Partial purification and biochemical characterization of a heteromeric protein phosphatase 2A holoenzyme from maize (Zea mays L.) leaves that dephosphorylates C4 phosophoenolpyruvate carboxylase. 1150 60

The cellular localization of the 35 kDa, low molecular mass acid metallophosphatase (LMW AcPase) from the frog (Rana esculenta) liver and its activity towards P-Ser and P-Tyr phosphorylated peptides were studied. This enzyme was localized to the cytoplasm of hepatocytes but did not appear in other cells of liver tissue (endothelium, macrophages, blood cells). This LMW AcPase does not display activity towards (32)P-phosphorylase a under conditions standard for the enzymes of PPP family. Proteins containing P-Ser: rabbit (32)P-phosphorylasea and phosvitin are hydrolysed only at acidic pH and are poor substrates for this enzyme. The frog AcPase is not inhibited by okadaic acid and F(-) ions, the Ser/Thr protein phosphatase inhibitors. Moreover, the frog enzyme does not cross-react with specific antisera directed against N-terminal fragment of human PP2A and C-terminal conserved fragment of the eukaryotic PP2A catalytic subunits. These results exclude LMW AcPase from belonging to Ser/Thr protein phosphatases: PP1c or PP2Ac. In addition to P-Tyr, this enzyme hydrolyses efficiently at acidic pH P-Tyr phosphorylated peptides (hirudin and gastrin fragments). K(m) value for the hirudin fragment (7.55 +/- 1.59 x 10(-6) M) is 2-3 orders of magnitude lower in comparison with other substrates tested. The enzyme is inhibited competitively by typical inhibitors of protein tyrosine phosphatases (PTPases): sodium orthovanadate, molybdate and tungstate. These results may suggest that the LMW AcPase of frog liver can act as PTPase in vivo. A different cellular localization and different response to inhibition by tetrahedral oxyanions (molybdate, vanadate and tungstate) provide further evidence that LMW AcPase of frog liver is distinct from the mammalian tartrate-resistant acid phosphatases.
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PMID:The 35 kDa acid metallophosphatase of the frog Rana esculenta liver: studies on its cellular localization and protein phosphatase activity. 1283 81

We have analyzed the proteomes of two human melanoma cell lines (A375 and 526), and of the human melanocytes, (FOM 78), by two-dimensional electrophoresis (2D-PAGE) and liquid chromatography - tandem mass spectrometry (LC-MS/MS). Our comparative proteomic analysis revealed that six proteins were over-expressed in both melanoma cell lines as compared to melanocytes: galectin-1, inosine-5'-monophosphate dehydrogenase 2, serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform, protein DJ-1, cyclophilin A and cofilin-1. We show, for the first time, that only specific isoforms of these molecules are over-expressed in melanoma. Different protein profiles were also found between each individual melanoma cell line and the melanocytes. s-Methyl-5-thioadenosine phosphorylase, ubiquitin and ribosomal protein S27 a precursor, the basic form of protein DJ-1, annexin a1, proliferation associated protein 2g4, isoform alfa-enolase of alfa-enolase, protein disulfide-isomerase precursor, and elongation factor 2 were more strongly expressed in A375 cells compared to melanocytes. In 526 cells, 60s acidic ribosomal protein p1 and calreticulin precursor were more highly expressed than in melanocytes. These molecular differences may help in better understanding melanoma development and its different responsiveness to therapies. The identified proteins could be exploited as biomarkers or therapeutic targets for melanoma.
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PMID:Characterization of human melanoma cell lines and melanocytes by proteome analysis. 2185 57