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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)

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.
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PMID:Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity. 23 39

The ATP-phosphohydrolase activity of extracts prepared from bovine spermatozoa flagella (BSFE), was characterized with respect to enzyme, substrate, activator ion and salt concentration, temperature dependence and time stability. BSFE required the presence of a divalent cation for activity: Mg++ or Ca++ could function as activator; Mn++, Zn++ and Cd++ could not. EDTA, but not EGTA, was inhibitory to enzymatic activity. Ca++ inhibited the Mg++ stimulated activity. ATP was dephosphorylated more rapidly than GTP greater than CTP greater than ITP, and ADP was dephosphorylated at 40% of the rate of ATP. The magnesium activated ATPase was stimulated by potassium and inhibited by sodium ions. Activation of BSFE ATP-phosphohydrolase was maximal in the presence of Mg++ and ATP in equimolar concentrations and K+ (0.05-0.3 M) at 30 degrees C. Although the enzymatic activity of the extract was found to decrease rapidly with time, it could be maintained for up to three days by the addition of 2-beta-mercaptoethanol to the bovine spermatozoa flagellar extracts.
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PMID:Characterization of the ATP-phosphohydrolase activity of bovine spermatozoa flagellar extracts. 23 27

The kinetic properties of the nonmitochondrial ATP-dependent Ca sequestering mechanism in disrupted nerve terminal (synaptosome) preparations have been investigated with radioactive tracer techniques; all solutions contained DNP, NaN3, and oligomycin, to block mitochondrial Ca uptake. The apparent half-saturation constant, KCa, for the nonmitochondrial Ca uptake is approximately 0.4 micrometer Ca; the Hill coefficient is approximately 1.6. Mg is also required for the Ca uptake, and the apparent KMg is approximately 80 micrometer. ATP and deoxy-ATP, but not CTP, GTP, ITP, UTP, ADP, or cyclic AMP, promote Ca uptake; the KATP, is approximately 10 micrometer. ATP analogs with blocked gamma-phosphate groups are unable to replace ATP. Particulate fractions from the disrupted synaptosomes possess Ca-dependent ATPase activity in the presence of Mg; the apparent KCa for this activity is 0.4--0.8 micrometer Ca, and the Hill coefficient is approximately 1.6. The Ca uptake and ATPase kinetic data suggest that the hydrolysis of 1 ATP may energize the transport of two Ca2+ ions into the storage vesicles. The second part of the article concerns the intraterminal distribution of Ca in "intact" terminals. When the terminals are disrupted after 45Ca loading, about one-half of the 45Ca is retained in the particulate material; some of this Ca, presumably stored in mitochondria, is released by the uncoupler, FCCP. Some of the 45Ca is released by A-23187, but not by FCCP; this fraction may be Ca stored in the nonmitochondrial sites described above. The proportion of 45Ca stored in the nonmitochondrial sites is increased when the Ca load is reduced or when the mitochondria are blocked with ruthenium red. These data indicate that the nonmitochondrial Ca storage sites are involved in intraterminal Ca buffering; they may play an important role in synaptic facilitation and post-tetanic potentiation, which result from Ca retention after neural activity.
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PMID:Calcium buffering in presynaptic nerve terminals. II. Kinetic properties of the nonmitochondrial Ca sequestration mechanism. 70 6

Hydrolysis of extracellular ATP and other nucleoside phosphates by A-431 human epidermoidal carcinoma cells was studied. The hydrolysis of extracellular ATP by these cells required either Mg2+ or Ca2+, and either cation could be replaced by Co2+, Fe2+, or Mn2+. Nucleoside triphosphates (ATP, GTP, CTP, UTP, and dTTP), but not nucleoside diphosphates, were hydrolyzed by the cells with Km and Vmax values similar to those for ATP (0.9-1.1 mmol/l and 6-10 nmol Pi formed/10(6) cells, respectively). The hydrolysis of ATP was inhibited strongly by ATP-gamma S and AMPPNP, and weakly by AMPCPP and ADP-beta S, but not by AMPCPP or AMPCP. Since the hydrolysis of [gamma-32P]ATP was inhibited by all these nucleoside triphosphates, the binding site for ATP is presumed to be the same as that for the other nucleoside triphosphates. All these results indicate that ecto-ATPase activity associated with A-431 cells is due to ecto-nucleoside triphosphatase. The nucleotide specificity shown in the present study indicates that ecto-nucleoside triphosphatase associated with A-431 cells is a molecule different from P2-purinergic receptors which can be stimulated specifically with nucleoside phosphates like ATP, ADP, UTP, UDP, and GTP, but not by other nucleotides.
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PMID:Characterization of ecto-nucleoside triphosphatase on A-431 human epidermoidal carcinoma cells. 129 31

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.
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PMID:Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity. 129 92

We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.
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PMID:A novel DNA helicase from calf thymus. 132 24

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

Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent Km = 0.40 +/- 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), could also stimulate efflux, but to a lesser extent. Also, ATP gamma S and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent Km = 46 +/- 7 microM), but extremely high capacity (106 +/- 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP-dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophthalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.
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PMID:Energy-dependent efflux of methotrexate in L1210 leukemia cells. Evidence for the role of an ATPase obtained with inside-out plasma membrane vesicles. 138 81

Fluorescein 5'-isothiocyanate (FITC) was used to modify the lysine residue in the active site of tonoplast H(+)-ATPase from etiolated mung-bean (Vigna radiata L.) seedlings. FITC caused marked inactivation of the enzyme activities of both membrane-bound and soluble ATPase and its associated H+ translocation. The SDS/PAGE pattern revealed that the FITC-binding site was in the large (A) subunit of ATPase. Inhibition could be substantially prevented by its physiological substrate ATP, pyrophosphate and nucleotides in the decreasing order: ATP greater than pyrophosphate greater than ADP greater than AMP greater than GTP greater than CTP greater than UTP. The mode of inhibition by FITC was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first-order kinetics with a Ki of 0.33 mM, a minimum inactivation half-time of 110 s, and a first-order rate constant of 0.244 s-1. A double-logarithmic plot of apparent rate constant versus FITC concentration gave a slope of 0.913, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labelling studies indicated that the incorporation of approx. 1 mol of FITC/mol of ATPase is sufficient to inhibit ATPase completely. The enhancement and blue shift of emission maxima of FITC after modification of ATPase indicated that the labelled lysine residue was located in a relatively hydrophobic domain.
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PMID:Inhibition of tonoplast ATPase from etiolated mung bean seedlings by fluorescein 5'-isothiocyanate. 138 33

The clpB gene in Escherichia coli encodes a heat-shock protein that is a close homolog of the clpA gene product. The latter is the ATPase subunit of the multimeric ATP-dependent protease Ti (Clp) in E. coli, which also contains the 21-kDa proteolytic subunit (ClpP). The clpB gene product has been purified to near homogeneity by DEAE-Sepharose and heparin-agarose column chromatographies. The purified ClpB consists of a major 93-kDa protein and a minor 79-kDa polypeptide as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Upon gel filtration on a Superose-6 column, it behaves as a 350-kDa protein. Thus, ClpB appears to be a tetrameric complex of the 93-kDa subunit. The purified ClpB has ATPase activity which is stimulated 5-10-fold by casein. It is also activated by insulin, but not by other proteins, including globin and denatured bovine serum albumin. ClpB cleaves adenosine 5'-(alpha,beta-methylene)-triphosphate as rapidly as ATP, but not adenosine 5'-(beta,gamma-methylene)-triphosphate. GTP, CTP, and UTP are hydrolyzed 15-25% as well as ATP. ADP strongly inhibits ATP hydrolysis with a Ki of 34 microM. ClpB has a Km for ATP of 1.1 mM, and casein increases its Vmax for ATP without affecting its Km. A Mg2+ concentration of 3 mM is necessary for half-maximal ATP hydrolysis. Mn2+ supports ATPase activity as well as Mg2+, and Ca2+ has about 20% their activity. Anti-ClpB antiserum does not cross-react with ClpA nor does anti-ClpA antiserum react with ClpB. In addition, ClpB cannot replace ClpA in supporting the casein-degrading activity of ClpP. Thus, ClpB is distinct from ClpA in its structural and biochemical properties despite the similarities in their sequences.
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PMID:The heat-shock protein ClpB in Escherichia coli is a protein-activated ATPase. 140 Mar 61


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