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)

An RNA helicase, isolated from nuclear extracts of HeLa cells, displaced duplex RNA in the presence of any one of the eight common nucleoside triphosphates. The unwinding reaction was supported most efficiently by ATP and GTP and poorly by dCTP and dTTP. The enzyme activity, purified 300-fold, contained two major protein bands of 80 and 55 kDa when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All fractions that contained RNA helicase activity also possessed single-stranded RNA-dependent nucleoside triphosphatase activity. Purified RNA helicase fractions displaced a hybrid of U4/U6 RNAs with the same efficiency as it displaced other duplex RNA structures. In contrast, the RNA helicase did not displace duplex RNA/DNA and DNA/DNA structures. Evidence is presented that suggests that this RNA helicase can displace duplex RNA by translocating in both the 3' to 5' and the 5' to 3' directions. The properties of the RNA helicase described here differ from the deaminase RNA unwinding activity described in Xenopus oocytes (Bass, B.L., and Weintraub, H. (1987) Cell 48, 607-613) and from the p68 HeLa RNA helicase (Hirling, H., Scheffner, M., Restle, T., and Stahl, H. (1989) Nature 339, 562-564).
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PMID:The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells. 170 30

Sarcolemmal membranes were isolated from skeletal muscle by a sucrose density gradient method from rats with diabetes induced by a streptozotocin injection (65 mg/kg iv). The activities of Na(+)-dependent Ca2+ uptake and Ca2(+)-stimulated adenosine-triphosphatase (ATPase) in the sarcolemmal fraction from diabetic rats was higher than those from the control animals. These changes were apparent at various times of incubation (1-10 min) as well as at different concentrations of free Ca2+ (10(-7) to 10(-5) M) and developed during the third and/or fourth weeks after streptozotocin injection. ATP-dependent Ca2+ uptake in the sarcolemmal vesicles was also increased at 28 and 56 days after inducing diabetes. Treatment of diabetic animals with insulin for 14 days reversed the changes in Ca2+ transport activities toward the control levels. Sarcolemmal Mg2(+)-ATPase and Na(+)-K(+)-ATPase activities remained unchanged in diabetic preparations. Furthermore, no difference in the sarcolemmal phospholipid composition and sodium dodecyl sulfate-gel electrophoretic pattern was evident between the control and experimental groups. These results indicate a higher activity of the sarcolemmal Ca2+ transport, which may be associated with hyperfunction of the skeletal muscle in diabetic rats.
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PMID:Increased sarcolemmal Ca2+ transport activity in skeletal muscle of diabetic rats. 185 Feb 3

Previous work suggested that the major Mr 46,000 ATP-binding protein [a putative nucleoside triphosphatase (NTPase)] found in rat liver nuclear scaffold (NS) may be proteolytically derived from lamins A/C. To definitively establish this identification, we undertook a series of photolabeling, proteolysis, and immunoprecipitation experiments. Mice were immunized with human lamin C expressed in bacteria, and monoclonal antibody-producing hybridomas were obtained. The purified monoclonal antibodies all recognized lamins A and C on immunoblots of NS, as well as Mr 46,000 or 34,000 proteolytic fragments as minor components. The Mr 46,000 photolabeled band was the only major NS component photolabeled with low concentrations of azido-ATP, and it was immunoprecipitated with anti-lamin monoclonal antibodies. To preclude the possibility that the photolabeled Mr 46,000 protein represented a minor component which comigrated with the Mr 46,000 lamin fragment and which specifically associated with lamins A/C during immunoprecipitation, a series of proteolytic digestions were undertaken. Digestion of the photolabeled Mr 46,000 peptide with chymotrypsin and staphylococcal protease V8 produced a limited number of photolabeled fragments, all of which comigrated with major stainable fragments produced from the Mr 46,000 lamin fragment. Cyanogen bromide cleavage of the photolabeled Mr 46,000 polypeptide, followed by polyacrylamide gel electrophoresis or high performance liquid chromatography/amino acid analyses, defined the COOH-terminal cleavage site as the Y residue at amino acid 376 and localized the photolabeled site to the COOH-terminal region (amino acids 372-376). In support of this proposed proteolytic cleavage site, specific assays with tyrosine-containing thiobenzyl ester substrate documented the presence of NS protease activity which cleaves at tyrosine residues; this activity shows a Km of 0.2 mM and a Kcat of approximately 250/s. Parallel experiments with mildly proteolyzed cloned lamin C preparations showed selective photolabeling of an Mr 34,000 fragment, which corresponds to a proteolytic breakdown product of the Mr 46,000 NS polypeptide; this Mr 34,000 photolabeled fragment was also immunoprecipitated with anti-lamin monoclonal antibodies and contained the same photolabeled site as the Mr 46,000 peptide. Cloned lamin C preparations were inactive in NTPase assays but did exhibit substantial ATP binding with an apparent KD = 4 x 10(-5) M ATP. These results indicate that the major Mr 46,000 photoaffinity-labeled protein in NS, which represents the putative NTPase thought to participate in nucleocytoplasmic transport, is derived from lamin A or lamin C by NS proteolytic activity which exposes a cryptic ATP-binding site near the highly conserved end of coil-2.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The Mr 46,000 nuclear scaffold ATP-binding protein: identification of the putative nucleoside triphosphatase by proteolysis and monoclonal antibodies directed against lamins A/C. 196 40

Addition of ATP or ATP analog to the incubation media is shown to result in cell death in experiments with different cultured cell lines as evidenced by the results of several independent assays, both in the absence or presence of extracellular Ca2+. Cytolytic T-lymphocyte (CTL) clone itself was not only resistant to cytolytic effects of ATP, but was able to "rescue" antigen-nonbearing 51Cr-labeled cells from lytic effects of extracellular ATP (but not from lytic effects of adenosine 5'-thiotriphosphate) when present during assay. To test whether the resistance of CTL to ATP is due to a high activity of ecto-ATPase, four independent assays of ATPase activity were utilized to demonstrate the presence and relatively high activity of the ecto-ATPase(s) on CTL surface. Studies of substrate specificity of CTL ecto-ATPase suggest that there is more than one nucleoside 5'-triphosphatase on the surface of CTL. The enzyme(s) activity is Ca2+- and Mg2+-dependent and in this respect is similar to recently described hepatic cells ecto-ATPase. We tested effects of known ATP-binding site-specific reagents fluorescein 5'-isothiocyanate (FITC) and 5'-fluorosulfonylbenzoyladenosine (FSBA) to find covalent modification procedures to be used in studies of functional role of ecto-ATPase. FSBA, but not FITC, inhibits lymphocyte ecto-ATPase but addition of ATP together with FSBA protects ecto-ATPase activity. Inactivation of CTL ecto-ATPase by pretreatment with FSBA makes CTL susceptible to lytic effects of extracellular ATP, as was hypothesized for the functional role of this enzyme in CTL.
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PMID:Ecto-ATPase activity in cytolytic T-lymphocytes. Protection from the cytolytic effects of extracellular ATP. 213 37

Chromobindin A is a large, multisubunit protein that binds to chromaffin granule membranes in a Ca2+- and ATP-regulated manner. Ca2+ stimulates binding to the membrane, whereas ATP, in the the absence of Ca2+, is required for release of the protein from the membrane. We now report that spectral and HPLC data indicate that nucleotides are associated with the native chromobindin A complex and that the protein can bind two molecules of [3H]ATP in vitro. Chromobindin A also appears to be a novel nucleotide triphosphatase. ATPase activity was detected in fractions containing chromobindin A isolated by affinity chromatography, gel filtration, or ion exchange chromatography. Kinetic studies indicated that the Vmax is 44 nmol of Pi/mg/min and the Km is 0.115 mM, whereas the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate acts as a competitive inhibitor of this reaction with a Ki of 0.08 mM. The activity was found to be sensitive to protease treatment or to preincubation at 65 degrees C and was inhibited by Ca2+ or low pH. The ATPase activity was not inhibited by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, vanadate, oligomycin, or azide.
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PMID:Interactions of the complex secretory vesicle binding protein chromobindin A with nucleotides. 213 61

Polyoma virus large tumor antigen (PyV T antigen) has been purified to near homogeneity by immunoaffinity column chromatography. We have detected DNA helicase and ATPase (nucleoside-5'-triphosphatase) activities in the purified PyV T antigen fraction and characterized these activities. The ATPase activity was stimulated about 2-fold by poly(dT), which was the most effective stimulator among the synthetic polynucleotides tested. Natural nucleic acids, such as calf thymus native and heat-denatured DNA, and single-stranded circular fd DNA were also effective, but the degree of stimulation was less than 1.5-fold. The basal and poly(dT)-stimulated ATPase activities showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optima. The preference for nucleoside 5'-triphosphates was ATP, dATP greater than CTP, UTP much greater than GTP. The only difference observed between the two activities was salt sensitivity. The basal ATPase activity was resistant to KC1 up to 300 mM. In contrast, poly-(dT)-stimulated activity was reduced to the level of basal activity at 300 mM KC1. DNA helicase activity required divalent cations and was dependent on hydrolysis of ATP. The activity showed similar preference for nucleoside 5'-triphosphates, requirement for divalent cations, and pH optimum as the two ATPase activities, and the salt sensitivity of DNA helicase activity was similar to that of poly(dT)-stimulated ATPase activity. The helicase activity was inhibited competitively by the addition of single-stranded or double-stranded DNA, and a relatively high inhibitory activity was observed with poly [d(A-T)]. The PyV T antigen helicase was found to migrate in the 3' to 5' direction along the DNA strand to which the protein bound.
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PMID:DNA helicase and nucleoside-5'-triphosphatase activities of polyoma virus large tumor antigen. 216 Feb 69

A photometric method for the determination of the acid nucleoside triphosphatase (EC 3.6.1.-) is described, in which inorganic phosphate is liberated from ATP or other nucleoside triphosphates. Colorimetric determination of liberated phosphate is based on the formation of a green complex of phosphomolybdate and malachite green hydrochloride. Optimal test conditions were evaluated as well as the sample preparation. The enzyme activities measured in 100 normal human sera are in the range of 0.5 to 9.0 U/l with an average of 4.0 U/l for men and 3.8 U/l for women.
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PMID:A colorimetric assay for the determination of acid nucleoside triphosphatase activity. 217 May 61

Evidence is presented for the existence of ectoenzymes in rat renal cortical brush-border membrane vesicles that produce adenosine as a final product using either ATP, ADP or AMP as substrate. The enzymes are insensitive to levamisole, ouabain, oligomycin and N-ethylmaleimide, and have absolute requirement for divalent cations with following order of activation Mg2+ greater than Ca2+ greater than Mn2+ greater than Ba2+ greater than Zn2+. At least two separate enzymes can be distinguished. One is capable of hydrolyzing ATP, other nucleoside triphosphates and ADP, but not AMP. The enzyme is insensitive to concanavalin A. The other enzyme hydrolyzes AMP and is strongly inhibited by this lectin. Mg2(+)-stimulated ATP hydrolysis displays saturation kinetics which is not of the simple Michaelis-Menten type, but is biphasic with a high-affinity (K'm = 0.16 mM) and low-affinity site (K'm = 9.0 mM), respectively. The low-affinity site hydrolyzes ATP, ITP and GTP to a similar extent, whereas CTP and UTP with about 40% lower rate. The high-affinity site splits ATP much better than other nucleoside triphosphates. Hydrolysis of ADP follows simple Michaelis-Menten saturation kinetic with apparent Km = 0.38 +/- 0.06 mM. Inhibition, activation and substrate specificity studies indicate that nucleoside triphosphatase and nucleoside diphosphatase may reside on the same protein. Kinetics of the AMP hydrolysis is hyperbolic with apparent Km = 76 +/- 9 microM. The cascade of ectonucleotidases in the brush-border membrane of the proximal tubule may catalyze the degradation of filtered nucleotides into adenosine and phosphate, the compounds which are thereafter probably reabsorbed by separate transport systems.
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PMID:The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranes. 217

Factors influencing the activity of the nucleoside analogue arabinosyl-5-azacytosine (ara-AC) were studied in P388 murine lymphoblasts in vitro and in vivo, in variants of these cells with artificially acquired resistance, in the naturally resistant colon 38 carcinoma in vivo, and in a panel of six human tumors maintained in continuous culture. Differences were noted not only between the sensitive and artificially developed resistant variants of P388, but also between the naturally sensitive (P388) and naturally resistant (colon 38) tumors. The artificially developed resistant P388 cell lines showed an inhibited capacity to accumulate nucleotides derived from ara-AC and deoxycytidine, whereas the accumulation of cytidine nucleotides remained unchanged. Studies of the initial velocity of facilitated diffusion of ara-AC showed only minor differences between parental and resistant lines, while the nucleotide formation rates from both ara-AC and deoxycytidine were markedly depressed in the latter cells. It is concluded, therefore, that the failure of resistant P388 cells to accumulate these compounds results not from a transport deficit per se but rather from a failure to convert the nucleosides to nondiffusible (i.e., phosphorylated) species inside the cell. This failure was accompanied by a substantial reduction in the incorporation of a radiolabeled product derived from deoxycytidine into the nucleic acids of the resistant clones. The common factor responsible for the resistance of P388 variants toward ara-AC appears to be a markedly decreased level of deoxycytidine kinase activity. The naturally resistant colon 38 carcinoma, on the other hand, in addition to a decrease in the activity of its deoxycytidine kinase, showed a lower level of activity of all its purine and pyrimidine kinases, along with a notably elevated nucleoside triphosphatase activity (with ATP as substrate) when compared to P388. These differences were reflected in lower endogenous nucleoside triphosphate pool sizes in colon 38, and in a lower level of ara-AC-5'-triphosphate accumulation in colon 38 than in P388 after comparable drug exposure. In the six human tumor lines, a positive correlation was established between sensitivity to ara-AC (as determined by its median inhibitory concentration) and cellular content of deoxycytidine kinase. It is concluded that this latter enzyme is a generally important determinant of sensitivity to arabinosyl-5-azacytosine.
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PMID:Arabinosyl-5-azacytosine: mechanisms of native and acquired resistance. 242 54

ATP-promoted efflux of poly(A)-rich RNA from isolated nuclei of prelabeled mouse lymphoma L5178y cells has an activation energy of 51.5 kJ/mol, similar to that found for the nuclear envelope nucleoside triphosphatase (48.1 kJ/mol) assumed to be involved in mediating nucleocytoplasmic transport of at least some RNA. Here we show that efflux of two specific poly(A)-rich mRNAs (actin and beta-tubulin) from isolated L-cell nuclei is almost totally dependent on the presence of ATP, while efflux of poly(A)-free histone mRNA (H4, H2B, and H1) also occurs to a marked extent in the absence of this nucleotide. Measurements of temperature dependence of transport rate revealed an activation energy of 56.1 kJ/mol for actin mRNA, while the activation energy for histone-H4-mRNA efflux was in the same range as that found for ATP-induced release of RNA from demembranated nuclei (about 15-20 kJ/mol). Addition of nonhydrolyzable nucleotide analogs of ATP to the in vitro system used for measurement of RNA transport did not result in release of nonhistone mRNA (actin), but enhanced the efflux of H4 mRNA to approximately the same extent as ATP. Although not absolutely required, addition of ATP stimulated the rate of export of histone mRNA about twofold. Only the poly(A)-rich RNA, but not the poly(A)-free RNA, released from isolated nuclei was found to compete with poly(A) for the nuclear envelope mRNA-binding site, indicating the mechanism of transport for both RNA classes to be distinct. Export of both nonhistone and histone mRNA was found to be inhibited by a monoclonal antibody against a p60 nuclear-pore-complex antigen. This antibody had no effect on the nucleoside triphosphatase, mediating transport of poly(A)-rich mRNA.
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PMID:Energy requirement and kinetics of transport of poly(A)-free histone mRNA compared to poly(A)-rich mRNA from isolated L-cell nuclei. 256 12


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