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)

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of Mg2+ and ATP. ADP, AMP, GTP, UTP, ITP, TTP, or CTP did not substitute for ATP in energizing uptake. The Vmax for Ca uptake was 2.4 pmol of Ca/micrograms of protein/min, and the Km values for Ca and ATP were 1.0 and 80 microM, respectively. One microM A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na+ or K+ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a Vmax of 25 pmol of Pi/micrograms of protein/min at a free Ca concentration of 4-5 microM. This Ca-ATPase had Km values for Ca and ATP of 0.6 and 90 microM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na+ or K+ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATPase activity of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivities to calmodulin.
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PMID:Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles. 645 67

The DNA-dependent ATPase activity of the recA protein of Escherichia coli shows a complex dependence on ATP concentration. With a single-stranded (SS) DNA cofactor, the Hill coefficient for ATP is 3.3 at pH 8.1 and 1.4 at pH 6.2. With a double-stranded (DS) DNA cofactor, the Hill coefficient is 3.3 at pH 6.2 (no reaction is detectable at pH 8.1). In the presence of SS DNA, the Km for ATP is 20 microM, independent of pH, while with DS DNA at pH 6.2, KmATP is 100 microM. These and other observations indicate that the interaction of recA protein with ATP is influenced by both pH and DNA cofactor. ADP, UTP, dTTP, and GTP are competitive inhibitors of the ATPase activity of recA protein, indicating that there is a single binding site for nucleoside triphosphates. Nucleoside triphosphates, but not ADP, reduce the Hill coefficient for ATP hydrolysis and thus can contribute to the cooperative effect of ATP.
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PMID:Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis. 645 29

The bacteriophage T7 gene 4 protein is a multifunctional enzyme that has DNA helicase, primase, and deoxyribonucleotide 5'-triphosphatase activities. Prior studies have shown that in the presence of dTTP or dTDP the gene 4 protein assembles into a functionally active hexamer prior to binding to single-stranded DNA. In this study, we have examined the effects of different nucleotide cofactors on the conformation of the gene 4 protein in the presence and absence of DNA. Gel retardation analysis, partial protease digestion, and DNA footprinting all suggest that the gene 4 protein undergoes a conformational change when dTTP is hydrolyzed to dTTP and that in the presence of dTDP the complex with DNA is more open or extended. We have also found that the dissociation constant of the gene 4 protein.DNA complex in the presence of dTDP was 10-fold lower than that determined in the presence of dTTP, further suggesting that these cofactors exerts different allosteric effects on the DNA-binding site of the gene 4 protein.
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PMID:Nucleotide and DNA-induced conformational changes in the bacteriophage T7 gene 4 protein. 759 68

Ecto-ATPase activity of Xenopus oocytes was studied by measuring the production of inorganic phosphate (Pi) from the breakdown of extracellular ATP. Enzyme activity involved Ca2+/Mg(2+)-dependent and Ca2+/Mg(2+)-independent dephosphorylation of ATP. Ca2+/Mg(2+)-dependent ecto-ATPase was active over a limited range of 0.01-1.0 mM ATP, while Ca2+/Mg(2+)-independent ATPase activity was active over a range of 0.1-30 mM ATP. Total enzyme activity was insensitive to changes in buffer pH (pH 7.0-9.0), but increased in a relatively linear manner with: (1) time of reaction (0-90 min), (2) number of cells (1-20 oocytes), and (3) temperature (10-37 degrees C). Ecto-ATPase activity was unaffected by ouabain (100 microM), sodium azide (100 microM), and oligomycin (5 micrograms/ml) (as inhibitors of endo-ATPases) and beta-glycerophosphate (10 mM) and p-nitrophenyl phosphate (10 mM) (as inhibitors of non-specific alkaline phosphatase). Total ecto-ATPase activity was reduced significantly in defolliculated oocytes, suggesting that the enzyme was located mainly on the enveloping follicle cell layer. The range order of preferential substrates was: ATP>GTP, ITP, UTP, CTP, TTP, 2-methylthioATP>ADP, 2-methylthioADP, AMP>>alpha, beta-methylene ATP, beta, gamma-methylene ATP, in the presence of divalent ions (where G is guanosine, I is inosine, U is uridine, C is cytidine and T is ribosylthymine). The P2-purinoceptor antagonist suramin [8-(3-benzamido-4-methylbenzamido)napthalene-1,3,5-trisul phonic acid), 100 microM] significantly inhibited total ecto-ATPase activity; this inhibition was competitive for the Ca2+/Mg(2+)-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characteristics of ecto-ATPase of Xenopus oocytes and the inhibitory actions of suramin on ATP breakdown. 892 22

Incubation of a glycoprotein fraction obtained from rat liver plasma membrane which has been previously well characterized using [gamma-32P]ATP results in the phosphorylation of a 230-kDa glycoprotein (pgp230). It is composed of a 120-kDa subunit (pgp120) and a 110-kDa subunit (pgp110) linked by interchain disulfide bonds. Peptide maps of pgp120 and pgp110 suggest extensive similarity in their polypeptide chains. Glycan analysis reveals between four and six hybrid-type oligosaccharide chains for both phosphoproteins. Immunoblotting using monoclonal antibodies and endoglycosidase digestion exclude an identity of pgp120 or pgp110 with the hepatocyte plasma membrane glycoproteins dipeptidylpeptidase IV or the taurocholate transport protein, which co-purify and co-migrate in SDS/PAGE. Protein phosphorylation is Ca(2+)-dependent (K0.5(Ca2+) = 0.35 microM, in the absence of Mg2+). In the presence of Mg2+, the glycoprotein undergoes rapid cycles of phosphorylation and dephosphorylation, resulting in ATPase activity. Analysis of phosphorylated amino acids identifies phosphothreonine as the major one. Photoaffinity labeling with 8-azido-[alpha-32P]ATP demonstrates the presence of one or more ATP binding site(s). Preincubation of pgp230 with various purine or pyrimidine nucleotides (ATP, UTP, TTP, ADP, GDP, AMP, CMP) or known P2-purinoceptor agonists or antagonists (adenosine 5'-[alpha,beta-methylene]triphosphate, 2-methyl-thio-adenosine 5'-triphosphate, suramin) inhibits its phosphorylation by [gamma-32P]ATP. The biological function of pgp230 is unknown at present. Several findings of the present study are compatible with the idea that pgp230 may be involved in a P2-purinoceptor function of the hepatocyte. Following this concept, a mechanism is discussed where a cytosolically exposed high-affinity Ca(2+)-binding site of pgp230 would allow for receptor feedback control, via phosphorylation and dephosphorylation, by sensing changes in cytosolic Ca2+ concentration.
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PMID:A new type of Ca(2+)-dependent, Mg(2+)-stimulated ATPase of rat liver plasma membrane. 781 88

1. Parotid plasma membrane nonpump low-affinity Ca(2+)-ATPase, which possesses high-affinity (Ca2+ + Mg2+)-ATPase activity, was characterized. 2. Purified Ca(2+)-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67-93% of ATP) and nucleoside diphosphates, ADP, GDP, IDP, CDP, TDP (12-40% of ATP) but not AMP and p-NPP. 3. The maximum activities of Ca(2+)- and (Ca2+ + Mg2+)-ATPases were obtained in the presence of 1 mM and 0.13 microM Ca2+, respectively. 4. The Km values for Ca2+ in Ca(2+)- and (Ca2+ + Mg2+)-ATPases were 0.2 mM and 22 nM, respectively. 5. The activities of both Ca(2+)- and (Ca2+ + Mg2+)-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction. 6. These features suggest that Ca(2+)-ATPase is an ecto-Ca(2+)-dependent nucleoside triphosphatase.
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PMID:The possibility that Ca(2+)-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca(2+)-dependent nucleoside triphosphatase. 806 15

A novel DNA helicase isolated from Xenopus laevis ovaries [Poll, E. H. A., & Benbow, R. M. (1988) Biochemistry 27, 8701-8706] was characterized biochemically. The directionality of DNA unwinding was determined to be 3' to 5'. A short 3' ssDNA tail adjacent to duplex DNA was required for DNA unwinding; the minimum length of this tail was between four and nine bases. Only short duplex DNA regions were unwound: duplex DNA of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not. Longer duplex regions were unwound in the presence of Escherichia coli single-strand DNA binding protein if, in addition, the duplex region was flanked by an unpaired 3' or 5' tail and the substrate resembled a branched replicative intermediate. X. laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA. DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 125 mM for Na chi PO4 or K chi PO4. The ATP analog ATP gamma S inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPCP and AMPPNP moderately inhibited DNA unwinding activity and had little effect on the copurifying DNA-dependent ATPase activity. CTP was a relatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dCTP, dGTP, or TTP showed moderate or no inhibition. The copurifying DNA-dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, dGTP, or TTP.
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PMID:Xenopus laevis ovarian DNA helicase I: A 3' to 5' helicase that unwinds short duplexes. 814 86

The ruvA and ruvB genes of Escherichia coli encode a novel DNA helicase that interacts with Holliday junctions and promotes branch migration. In this work, we have investigated the protein-DNA complexes formed between RuvA, RuvB and Holliday junctions. As shown previously, RuvA protein binds a synthetic Holliday junction in vitro, to form a specific protein-DNA complex that can be detected by a band-shift assay. We now show that the combined presence of RuvA and RuvB results in a super-shift of this complex indicative of the formation of a RuvAB-Holliday junction complex. In the absence of RuvA, the RuvB protein fails to bind Holliday junctions. The RuvAB-Holliday junction complex was detected by the band-shift assay only under conditions that favoured its stability, e.g. complex formation in the presence of a nucleoside triphosphate that can not be hydrolysed by RuvB (adenosine 5'-[gamma-thio]triphosphate). In contrast, nucleoside triphosphates that can be hydrolysed (ATP, dATP, dCTP or TTP), lead to RuvAB-mediated branch migration of the junction. These results indicate that the formation of a (RuvAB-ATP)-Holliday junction complex represents the first step in the process of branch migration, and that branch migration is dependent upon ATP hydrolysis. In addition, we show that Holliday junction DNA stimulates the ATPase activity of RuvAB to a greater extent than either single-stranded or linear duplex DNA.
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PMID:Formation of a RuvAB-Holliday junction complex in vitro. 839 34

DNA polymerases alpha, delta and epsilon from normal regenerating rat liver and Novikoff hepatoma cells were purified about 300-fold, characterized, and checked for sensitivity towards drugs known to inhibit cell proliferation. Characterization included (a) identification of associated proteins, (b) measurement of physiochemical constants (including sedimentation coefficients, diffusion coefficients, calculation of relative molecular masses), (c) quantification of catalytic activities using specific DNA primer templates (Km values) and specific inhibitors (Ki values), and (d) discrimination between DNA polymerases from normal cells and those from malignant cells using inhibitors of cell proliferation. (a) DNA primase associated with DNA polymerase alpha, and 3'-5' exonuclease accompanying DNA polymerases delta and epsilon had similar activities. (b) Comparison of physicochemical and catalytic properties of DNA polymerases from both sources revealed similarities but also some important differences. Sedimentation and diffusion coefficients of DNA polymerases alpha and epsilon from malignant cells differed significantly. (c) The DNA-binding domain of DNA polymerases alpha and epsilon from hepatoma cells was altered since Km values, determined with several specific DNA primer-templates, were higher. Furthermore, dNTP-binding sites of DNA polymerases from malignant cells, when probed with specific inhibitors (aphidicolin, butylphenyl-dGTP, carbonyldiphosphonate, and dideoxy-TTP) showed significantly lower Ki values, indicating lower affinity to deoxyribonucleoside 5'-triphosphates. (d) Sixteen drugs representative of various modes of interaction with DNA and protein were chosen. Dose/response experiments were performed and the concentration at which the polymerizing activity was reduced to 50% was calculated (K50 values). Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells was found for: the intercalating drugs doxorubicin, daunorubicin, amsacrine, mitoxantrone, quinacrine and ethidium bromide, the minor-groove binders distamycin and netropsin, the ATPase-blocking agents novobiocin and coumamycin, and the topoisomerase I inhibitors camptothecin and topotecan. When the sensitivity of polymerases delta and epsilon was measured using poly(dA.dT) as a primer-template, the preferential inhibition of the enzymes from malignant cells was even more pronounced. Drugs known to trap the DNA-topoisomerase-II complex, etoposide, nalidixic acid, teniposide, and merbarone did not affect DNA polymerases irrespective of the source. Since the majority of the inhibitors used, particularly intercalators and minor-groove binders, act by modification of the primer-template, inhibition of DNA synthesis must have occurred through weakening of non-covalent bonds between DNA and catalytic polypeptides. Consequently, preferential inhibition of DNA polymerases from malignant cells seems to be indicative of abnormally diminished binding of the enzymes to their primer-templates. This effect may be caused by conformational alterations in polymerases from malignant cells which affect the DNA binding domains. Similarly, changes in physicochemical and kinetic constants are indicative of alterations of dNTP-binding domains.
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PMID:Preferential inhibition of DNA polymerases alpha, delta, and epsilon from Novikoff hepatoma cells by inhibitors of cell proliferation. 857 84

Hepatocyte plasma membranes contain a glycosylated 230-kDa Ca(2+) -dependent, Mg(2+)-stimulated ATPase (pgp230), which consists of two subunits, one of 120 kDa and the other of 110 kDa. pgp230 can be enriched by the use of affinity chromatography on Concanavalin A-Sepharose, wheat germ lectin-Sepharose, and 5'-AMP-Sepharose. It has a high-affinity Ca2+ binding site. In the presence of Ca2+, it forms a phosphorylated intermediate by autocatalytic transfer of the terminal phosphate residue from ATP. Maximal Ca(2+)-dependent autophosphorylation is observed at pH 5-6. Photoaffinity labeling using 8-azido-[alpha-32P]ATP or [y-32P]ATP confirms the presence of ATP binding sites. Incubation with [alpha-32P]ATP leads to a rapid but transient labeling of pgp230. Various nucleotides, nucleotide receptor agonists, or antagonists inhibit Ca(2+)-dependent phosphorylation by [y-32P]ATP. The concentrations of half-maximal inhibition range from 10(-7) M to 10(-3) M. The rank order of inhibitory potency is: ATP > alpha,beta-methylene-ATP > CTP = TTP > y-4-amino-phenyl-ATP = 2-methyl-thio-ATP > UTP = GTP > GDP = ADP = beta,y-methylene-ATP = beta, y-methylene-TTP = beta,y-methylene-GTP = adenosine-5'-O-2-thiodiphosphate = CMP = AMP > adenosine > cytidine > guanosine = suramin > Reactive blue 2 > iso-butyl-methyl-xanthine > thymidine > uridine. These data suggest a nucleotide binding capacity of this new hepatocyte membrane glycoprotein. Further investigations should be carried out to reveal its biological function.
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PMID:Partial characterization of a new nucleotide binding glycoprotein of hepatocyte plasma membrane. 878 41


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