Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.25 (triphosphatase)
1,529 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chicken gizzard smooth muscle extracellular ATPase (ecto-ATPase) is a low abundance, high specific activity, divalent cation-dependent, nonspecific nucleotide triphosphatase (NTPase). The ATPase is a 66-kDa glycoprotein with a protein core of 53 kDa (Stout, J.G. and Kirley, T.L. (1994) J. Biochem. Biophys. Methods 29, 61-75). In this study we evaluated the characteristics of a bank of monoclonal antibodies raised against a partially purified chicken gizzard ecto-ATPase. 18 monoclonal antibodies identified by an ATPase capture assay were tested for effects on ATPase activity as well as for their Western blot and immunoprecipitation potential. The five most promising monoclonal antibodies were used to immunopurify the ecto-ATPase. The one-step immunoaffinity purification of solubilized chicken gizzard membranes with all five of these monoclonal antibodies isolated a 66-kDa protein whose identity was confirmed by N-terminal sequence analysis to be the ecto-ATPase. Several of these monoclonal antibodies stimulated ecto-ATPase activity similar to that observed previously with lectins. Western blot analysis revealed that three of the five monoclonal antibodies recognized a major immunoreactive band at 66 kDa (53-kDa core protein), consistent with previous purification results. The other two antibodies recognized proteins of approximately 90 and 160 kDa on Western blots. The 90-kDa co-immunopurifying (and presumably associated or related) protein was identified by N-terminal analysis as LEP100, a glycoprotein that shuttles between the plasma and lysosomal membranes. The approximately 160-kDa co-immunopurifying protein was identified by N-terminal analysis as integrin, a protein involved in extracellular contacts with adhesion molecules. Extended N-terminal sequence analysis of the immunopurified 66-kDa ecto-ATPase revealed some sequence homology with mouse lysosomal associated membrane protein. Tissue distribution of the ecto-ATPase showed that the highest levels of protein were expressed in muscle tissues (cardiac, skeletal, and smooth) and brain.
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PMID:Properties of and proteins associated with the extracellular ATPase of chicken gizzard smooth muscle. A monoclonal antibody study. 774 34

We have examined the RNA-capping enzyme activities of bluetongue virus (BTV) minor core protein, VP4. Recombinant BTV VP4 protein was purified to homogeneity from insect cell culture infected with a baculovirus VP4 of BTV serotype 10. We demonstrate that the purified protein, and VP4 encapsidated in core-like particles, react with GTP and covalently bind GMP via a phosphoamide linkage, a characteristic feature of guanylyltransferase enzyme. VP4 also catalyses a GTP-PPi exchange reaction indicating that the protein is the guanylyltransferase of the virus. In addition, VP4 possesses an RNA 5'-triphosphatase activity which catalyses the first step in the RNA-capping sequence. Further, an inorganic pyrophosphatase activity was identified which may aid the transcription activity within the virus by removing inorganic pyrophosphate which is an inhibitor of the polymerization reaction. Finally, the direct evidence of VP4 capping activity has been obtained by demonstrating in vitro transfer of GMP to the 5' end of in vitro synthesized BTV ssRNA transcripts to form a cap structure.
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PMID:Guanylyltransferase and RNA 5'-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus. 967 55

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein's interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
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PMID:Hepatitis C virus core protein interacts with cellular putative RNA helicase. 1007 32

To complement evidence for nucleoside triphosphate phosphohydrolase (NTPase), RNA helicase, RNA 5' triphosphate phosphohydrolase, and nucleic acid-binding activities by the core shell protein lambda1 of mammalian orthoreoviruses (reoviruses), we determined nucleotide sequences of the lambda1-encoding L3 gene segments from three isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). The T1L and T3D L3 gene sequences and deduced lambda1 protein sequences shared high levels of identity (97.7% and 99.3%, respectively). The lambda1 sequences differed at only 9 of 1275 amino acids. Two single-nucleotide insertions relative to a previously published sequence for T3D L3 extended the lambda1 open reading frame to within 60 nucleotides of the plus-strand 3' end for T3D and the other isolates sequenced, in keeping with the short 3' nontranslated regions of the other nine gene segments. Seven of the nine amino acid differences between T1L and T3D lambda1 were located within the amino-terminal 500 residues of lambda1, a region with putative sequence similarities to NTPases and RNA helicases. The T2J L3 and lambda1 sequences were found to be more divergent, especially within the amino-terminal 180 residues of lambda1, preceding the putative CCHH zinc finger motif. The T2J L3 sequence, along with partial sequences for L3 genes from three other reovirus isolates, suggested that one or more of the polymorphisms at amino acids 71, 215, 500, 1011, and/or 1100 in lambda1 contribute to the L3-determined differences in ATPase activities by T1L and T3D cores. The findings contribute to our ongoing efforts to elucidate sequence-structure-function relationships for the lambda1 core protein.
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PMID:Mammalian reovirus L3 gene sequences and evidence for a distinct amino-terminal region of the lambda1 protein. 1032 67

Analysis of the amino acid sequence of core protein muA of avian reovirus has indicated that it may share similar functions to protein mu2 of mammalian reovirus. Since mu2 displayed both nucleotide triphosphatase (NTPase) and RNA triphosphatase (RTPase) activities, the purified recombinant muA ( muA) was designed and used to test these activities. muA was thus expressed in bacteria with a 4.5 kDa fusion peptide and six His tags at its N terminus. Results indicated that muA possessed NTPase activity that enabled the protein to hydrolyse the beta-gamma phosphoanhydride bond of all four NTPs, since NDPs were the only radiolabelled products observed. The substrate preference was ATP>CTP>GTP>UTP, based on the estimated k(cat) values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide-binding site of muA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein muA. The activity of muA is dependent on the divalent cations Mg(2+) or Mn(2+), but not Ca(2+) or Zn(2+). Optimal NTPase activity of muA was achieved between pH 5.5 and 6.0. In addition, muA enzymic activity increased with temperature up to 40 degrees C and was almost totally inhibited at temperatures higher than 55 degrees C. Tests of phosphate release from RNA substrates with muA or K408A/K412A muA indicated that muA, but not K408A/K412A muA, displayed RTPase activity. The results suggested that both NTPase and RTPase activities of muA might be carried out at the same active site, and that protein muA could play important roles during viral RNA synthesis.
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PMID:Avian reovirus core protein muA expressed in Escherichia coli possesses both NTPase and RTPase activities. 1748 41

A minor core protein, VP6, of bluetongue virus (BTV) possesses nucleoside triphosphatase, RNA binding, and helicase activities. Although the enzymatic functions of VP6 have been documented in vitro using purified protein, its definitive role in BTV replication remains unclear. In this study, using a recently developed T7 transcript-based reverse genetics system for BTV, we examined the importance of VP6 in virus replication. We show that VP6 is active early in replication, consistent with a role as part of the transcriptase or packaging complex, and that its action can be provided in trans by a newly developed complementary cell line. Furthermore, the genomic segment encoding VP6 was mutated to reveal the cis-acting sequences required for replication or packaging, which subsequently enabled the construction of a chimeric BTV expressing enhanced green fluorescent protein. These data confirm that one of the 10 genome segments of BTV can be replaced with a chimeric RNA containing the essential packaging and replication signals of BTV and the coding sequence of a foreign gene.
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PMID:Bluetongue virus VP6 acts early in the replication cycle and can form the basis of chimeric virus formation. 1955 29

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter's binding to RhoGDI1, which in turn enhances RhoGDI1's affinity for RhoG. S4 clustering activates PKCalpha, which phosphorylates RhoGDI1 at Ser(96). This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4-synectin-RhoGDI1 protein complex and activation via PKCalpha.
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PMID:Suppression of RhoG activity is mediated by a syndecan 4-synectin-RhoGDI1 complex and is reversed by PKCalpha in a Rac1 activation pathway. 1958 9

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