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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An initial step in the replication of simian virus (SV40) DNA is the ATP-dependent formation of a double hexamer of the SV40 large tumor (T) antigen at the SV40 DNA replication origin. In the absence of DNA, T antigen assembled into hexamers in the presence of magnesium and ATP. Hexameric T antigen was stable and could be isolated by glycerol gradient centrifugation. The ATPase activities of hexameric and monomeric T antigen isolated from parallel glycerol gradients were identical. However, while monomeric T antigen was active in the ATP-dependent binding, untwisting, unwinding, and replication of SV40 origin-containing DNA, hexameric T antigen was inactive in these reactions. Isolated hexamers incubated at 37 degrees C in the presence of ATP remained intact, but dissociated into monomers when incubated at 37 degrees C in the absence of ATP. This dissociation restored the activity of these preparations in the DNA replication reaction, indicating that hexameric T antigen is not permanently inactivated but merely assembled into a nonproductive structure. We propose that the two hexamers of T antigen at the SV40 origin assemble around the DNA from monomer T antigen in solution. This complex untwists the DNA at the origin, melting specific DNA sequences. The resulting single-stranded regions may be utilized by the T antigen helicase activity to initiate DNA unwinding bidirectionally from the origin.
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PMID:The simian virus 40 T antigen double hexamer assembles around the DNA at the replication origin. 132 Nov 35

This study evaluated the effect of L-1-oleoyl-2-acetyl-sn-3-glycerol (OAG) on ouabain-sensitive Na,K-dependent oxygen consumption (Na,K-QO2) in intact renal proximal tubule cells (RPTC). Basal Na,K-QO2 (nmol O2/mg protein per min) was 20.0 +/- 1.0. Incubation with 10 nM of OAG induced a dual effect on Na,K-QO2, with an initial stimulation (maximal at 10 min, 37.1 +/- 5.0), followed by an inhibition (significant at 20 min, 16.3 +/- 1.0). No changes in ouabain-insensitive QO2 were observed in any of the protocols. The effects were abolished by sphingosine, a protein kinase C inhibitor. Stimulation was abolished by amiloride 0.1 mM. Amiloride had no effect on Na,K-QO2 at the concentration used. Stimulation was not potentiated by the sodium ionophore, amphotericin B, and the later inhibition was still observed in the presence of amphotericin B. The initial stimulation was attributed to an increase in sodium permeability, while the later inhibition was attributed to a direct effect on the Na,K-pump. Regulation of Na+,K(+)-ATPase activity by protein kinase C in intact RPTC can be accomplished by a direct effect on the protein or as a secondary effect consequent upon changes in intracellular sodium.
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PMID:Diacylglycerol activation of protein kinase C results in a dual effect on Na+,K(+)-ATPase activity from intact renal proximal tubule cells. 132 Nov 61

The regulation of the guinea-pig pancreatic acinar plasma membrane Ca2+ pump by protein kinase A, protein kinase C and calmodulin was investigated. The results were compared with the effects of these regulators on the high affinity Ca(2+)-ATPase found in this membrane preparation. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca2+ transport 2-fold, but had no effect on Ca(2+)-dependent ATPase activity. Purified protein kinase C, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate and diacylglycerol derivative, 1-stearoyl-2-arachidonoyl-sn-glycerol, failed to stimulate the Ca(2+)-uptake but augmented the Ca(2+)-dependent ATPase activity. Exogenously added calmodulin failed to stimulate either activity. In addition, two antagonists of calmodulin activity, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca(2+)-transport. These data suggest the presence of endogenous calmodulin within guinea-pig pancreatic acinar plasma membranes. Both calmodulin antagonists failed to influence the Ca(2+)-dependent ATPase activity. The ability of boiled extracts from guinea-pig pancreatic acinar plasma membranes to stimulate the Ca(2+)-ATPase activity in calmodulin-depleted erythrocyte plasma membranes confirmed the presence of endogenous calmodulin. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca2+ efflux from pancreatic acinar cells. These results also provide further evidence suggesting that the high affinity Ca(2+)-ATPase does not catalyze the plasma membrane Ca(2+)-transport activity observed in pancreatic acini.
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PMID:Regulation of calcium transport in pancreatic acinar plasma membranes from guinea pig. 132 90

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.
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PMID:A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. 132 72

At least six DNA helicases have been identified during fractionation of extracts from the yeast Saccharomyces cerevisiae. Three of those, DNA helicases B, C, and D, have been further purified and characterized. DNA helicases B and C co-purified with DNA polymerase delta through several chromatographic steps, but were separated from the polymerase by hydrophobic chromatography. DNA helicase D co-purified with Replication Factor C over seven chromatographic steps, and was only separated from it by glycerol gradient centrifugation in the presence of 0.2 M NaCl. All three helicases are DNA dependent ATPases with Km values for ATP of 190 microM, 325 microM, and 60 microM for DNA helicases B, C, and D, respectively. Their DNA helicase activities are comparable. They are 5'-3' helicases and have pH optima of 6.5-7 and Mg2+ optima of 1-2 mM. However, they differ in the nucleotide requirement for helicase action. Whereas all three helicases preferred ATP, dATP, UTP, CTP, and dCTP as cofactors, DNA helicase C also used GTP, but not dTTP. On the other hand, DNA helicase D used dTTP, but not GTP, and DNA helicase B used neither nucleotide as cofactor. These studies allowed us to conclude that DNA helicases B, C, and D are not only distinct enzymes, but also different from two previously identified yeast DNA helicases, the RAD3 protein and ATPase III.
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PMID:Three new DNA helicases from Saccharomyces cerevisiae. 133 84

Multidrug-resistant human tumor cells overexpress the MDR1 gene product P-glycoprotein, which is believed to function as an ATP-dependent efflux pump. In this study we demonstrate that the partially purified P-glycoprotein, when reconstituted in an artificial membrane, catalyzes drug-stimulated ATP hydrolysis. Plasma membrane proteins of a human multidrug-resistant cell line, KB-V1, were solubilized with 1.4% (wt/vol) octyl beta-D-glucopyranoside in the presence of 0.4% phospholipid and 20% (vol/vol) glycerol, and the crude detergent extract was chromatographed on DEAE-Sepharose CL-6B. The 0.1 M NaCl fraction, enriched in P-glycoprotein but devoid of Na,K-ATPase, was reconstituted by the detergent-dilution method. P-glycoprotein constituted 25-30% of the reconstituted protein in proteoliposomes. ATP hydrolysis by proteoliposomes was stimulated 3.5-fold by the addition of vinblastine but was unaffected by the hydrophobic antitumor agent camptothecin, which is not transported by P-glycoprotein. The stimulatory effect of vinblastine was observed only if the protein was reconstituted in proteoliposomes, suggesting that either the substrate binding site(s) was masked by detergent or that the conformation of the soluble P-glycoprotein might not be suitable for substrate-induced activation. Several other drugs that are known to be transported by P-glycoprotein enhanced the ATPase activity in a dose-dependent manner with relative potencies as follows: doxorubicin = vinblastine greater than daunomycin greater than actinomycin D greater than verapamil greater than colchicine. The basal and vinblastine-stimulated ATPase activities were inhibited by vanadate (50% inhibition observed at 7-10 microM) but were not affected by agents that inhibit other ATPases and phosphatases. These data indicate that the P-glycoprotein, similar to other ion-transporting ATPases, exhibits a high level of ATP hydrolysis (5-12 mumol per min per mg of protein).
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PMID:Partial purification and reconstitution of the human multidrug-resistance pump: characterization of the drug-stimulatable ATP hydrolysis. 135 64

The topographic location of the region comprising amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase has been elucidated using reconstituted proteoliposomes and protein chemical techniques. Proteoliposomes containing H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were cleaved with trypsin and the resulting digest was subjected to centrifugation on a glycerol step gradient to separate the released and liposome-bound peptides. The released peptides were recovered in the upper regions of the step gradient, whereas the liposome-bound peptides were recovered near the 40% glycerol interface. The released peptides present in the upper fractions were reduced, 14C-carboxy-methylated, and then separated by high performance liquid chromatography. Two radioactive cysteine-containing peptides with retention times of about 162 and 182 min were identified as H(+)-ATPase peptides comprising residues Leu363-Lys379 and Leu388-Arg414, respectively, by comparison to standards prepared from the purified ATPase. This information thus establishes a cytoplasmic location for residues 359-418 in the H(+)-ATPase polypeptide chain. It also infers a cytoplasmic location for residues 419-440, since this stretch of amino acids is too short to cross the membrane and return between regions known to be cytoplasmically located. These results and the results of other recent experiments establish the topographical location of nearly all of the 919 residues in the H(+)-ATPase molecule.
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PMID:Cytoplasmic location of amino acids 359-440 of the Neurospora crassa plasma membrane H(+)-ATPase. 138 55

The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.
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PMID:Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. 139 Aug 24

We have isolated mutants of the yeast Saccharomyces cerevisiae that are defective in localization of nuclear proteins. Chimeric proteins containing the nuclear localization sequence from SV40 large T-antigen fused to the N-terminus of the mitochondrial F1 beta-ATPase are localized to the nucleus. Npl (nuclear protein localization) mutants were isolated by their ability to grow on glycerol as a consequence of no longer exclusively targeting SV40-F1 beta-ATPase to the nucleus. All mutants with defects in localization of nucleolar proteins and histones are temperature sensitive for growth at 36 degrees C. Seven alleles of NPL3 and single alleles of several additional genes were isolated. NPL3 mutants were studied in detail. NPL3 encodes a nuclear protein with an RNA recognition motif and similarities to a family of proteins involved in RNA metabolism. Our genetic analysis indicates that NPL3 is essential for normal cell growth; cells lacking NPL3 are temperature sensitive for growth but do not exhibit a defect in localization of nuclear proteins. Taken together, these results indicate that the mutant forms of Npl3 protein isolated by this procedure are interfering with nuclear protein uptake in a general manner.
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PMID:A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast. 139 78

A novel DNA helicase has been isolated from Saccharomyces cerevisiae. This DNA helicase co-purified with replication factor C (RF-C) during chromatography on S-Sepharose, DEAE-silica gel high performance liquid chromatography (HPLC), Affi-Gel Blue-agarose, heparin-agarose, single-stranded DNA-cellulose, fast protein liquid chromatography MonoS, and hydroxyapatite HPLC. Surprisingly, the helicase could be separated from RF-C by sedimentation on a glycerol gradient in the presence of 200 mM NaCl. The helicase is probably a homodimer of a 60-kDa polypeptide, which by UV cross-linking has been shown to bind ATP. It has a single-stranded DNA-dependent ATPase activity, with a Km for ATP of 60 microM. The DNA helicase activity depends on the hydrolysis of NTP (dNTP), with ATP and dATP the most efficient cofactors, followed by CTP and dCTP. The DNA helicase has a 5' to 3' directionality and is only marginally stimulated by coating the single-stranded DNA with the yeast single-stranded DNA-binding protein RF-A.
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PMID:A Saccharomyces cerevisiae DNA helicase associated with replication factor C. 146 28


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