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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)

A conformational transition between E2 and E1 forms of Na, K-ATPase induced by different nucleotides has been studied under steady state conditions using the enzyme labelled with 5-iodoacetamidofluorescein. In the presence of K+ the plot of fluorescence as a function of [ATP], [ADP] or [CTP] (in a range of 5 microM-12 mM) is a biphasic one. A similar dependence for AMP, ITP, GTP and UTP demonstrates a hyperbolic behaviour. The data suggest that the shift in the equilibrium between E2 and E1 forms of Na,K-ATPase towards the E1 conformation is induced by ATP binding both with high and low affinity sites. Two structural features of ATP are apparently important for its interaction with more than one type of ATP binding sites or for providing for E2-E1 transition induced by this interaction: (i) beta-phosphate group in the terminal part of the molecule, (ii) unprotonated N1 and/or NH2-group in the 6th position of the purine base.
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PMID:Na,K-ATPase labelled with 5-iodoacetamidofluorescein: E2-E1 conformational transition induced by different nucleotides. 216 90

A well-characterized chicken osteoclast plasma membrane vesicle preparation manifested Mg2(+)-dependent ATP hydrolyzing activity of 0.213 mumol inorganic phosphate released per mg protein per minute (n = 7). The Mg2+ dependence showed a high-affinity component with a KMg of 1.293 microM and Vmax of 0.063 mumol Pi per mg protein per minute, and a low-affinity component with a KMg of 297.6 microM and a Vmax of 0.232 mumol Pi per mg protein per minute. The Mg2(+)-ATPase activity was inhibited by N,N'-dicyclohexylcarbodiimide (DCCD, 0.2 mM, 50.7%), N-ethylmaleimide (0.5 mM, 34.6%), nolinium bromide (1 mM, 29.9%), 4,4'-diisothiocyano-2,2'-stilbene sulfonic acid (DIDS, 1 mM, 45.1%), and p-chloromercuribenzoic acid (PCMB, 0.1 mM, 33.8%). Sodium orthovanadate (Na3 VO4) at 1 microM had no effect but caused 29.5% inhibition at 1 mM. Na+ could substitute for K+ without loss of activity, NO3- caused 19.5% inhibition when substituted for Cl-, and acetate replacement of Cl- resulted in 36.4% stimulation of Mg2(+)-ATPase. ATP, GTP, ITP, CTP, and ADP were all hydrolyzed effectively. DCCD (0.2 mM), NEM (0.5 mM), nolinium bromide (1 mM), and DIDS (50 microM) almost completely abolished proton transport as measured spectrofluorometrically by acridine orange quenching. Na3 VO4 (1 mM) had no effect, and duramycin (80 micrograms/ml) inhibited transport 52.7%. K+ replacement of Na+ caused a 79.2% increase in initial proton transport rate. NO3- and acetate substitution of Cl- resulted in a 46.1 and 55.7% decrease in transport, respectively. ATP supports transport far more effectively than the other nucleotides tested. ADP was ineffective. Experiments using the potassium ionophore, valinomycin, indicated that the proton pump functions electrogenically, with Cl- most likely cotransported by an anion transporter. The proton pump also seems to have at least one anion-sensitive site, elucidated by experiments in the presence of NO3- and Cl-.
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PMID:Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles. 216 21

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle. 216 18

To help characterize the Na,K-ATPase active site with enzyme incorporated into phospholipid vesicles, the activities with alternative substrates were compared, 22Na/Na-transport was equivalent with ATP, CTP, carbamylphosphate and acetylphosphate, but slower with CTP, 3-O-methylfluoresceinphosphate (3-O-MFP), nitrophenylphosphate and umbelliferonephosphate. It indicates a slower rate of formation of phosphorylating enzyme complex in conformation position of E1 (E1P) when the second group of substrates is bound with enzyme active center. 22Na/K-transport was half as effective with CTP as with ATP and was far slower with the other substrates. It indicates a more stringent selectivity at the low-affinity site of enzyme in conformation E2 that accelerates the slow step of this transport mode. Although enzyme modification with fluoresceinisothiocyanate blocks the high-affinity site to ATP, the K-phosphatase reaction catalyzed by E2 is retained, even with a substrate, 3-O-MFP, that binds to the adenine pocket. Dimethylsulfoxide inhibits hydrolysis of the nucleotides and of the carboxylic phosphate substrates of the K-phosphatase reaction, but stimulates hydrolysis of the phenolic phosphate substrates (nitrophenylphosphate and umbelliferone phosphate) which normally are hydrolyzed more slowly than the other substrates. On the basis of these data the authors propose the model of Na,K-ATPase active center.
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PMID:[The reaction mechanism of Na, K-ATPase]. 216 14

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

Approximately one-third of the total ATP-hydrolysis activity in isolated HeLa nuclei is sensitive to RNAase (ribonuclease). This activity is selectively extracted with pulse-labelled RNA. In the extracts it co-sediments with various particles with sedimentation coefficients from 10S to 50S, but especially with 24S and 40S particles. ATP hydrolysis by the isolated particles was inhibited extensively (greater than 80%) by RNAase A, heparin and 0.2 M-NaCl. The activity of RNAase-treated particles was recovered when poly(A) was added, but not when DNA was added. The isolated particles exhibited RNAase-sensitive hydrolysis activities for dATP, GTP, CTP and UTP as well as for ATP, and the UTPase activity in the extracts showed nearly the same sedimentation distribution as the ATPase activity. When samples of isolated particles were irradiated with u.v. light in the presence of [alpha-32P]ATP, a 39 kDa polypeptide with a broad distribution from 10S to 50S like that of the ATPase and a 55 kDa polypeptide with a sharp distribution at 24S were photolabelled. Taken together, the data suggest that ATP-hydrolysis activity found in nuclear ribonucleoprotein subfractions appears to be the result of one or two RNA-dependent NTPases that are normally associated with endogenous RNA in a wide variety of particles.
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PMID:Characterization of a ribonuclease-sensitive nucleoside triphosphatase activity from HeLa nuclei. 240 2

An ATP-dependent calcium transport component from rat liver plasma membranes was solubilized by cholate and reconstituted into egg lecithin vesicles by a cholate dialysis procedure. The uptake of Ca2+ into the reconstituted vesicles was ATP-dependent and the trapped Ca2+ could be released by A23187. Nucleotides, including ADP, UTP, GTP, CTP, GDP, AMP, and adenyl-5'-yl beta, gamma-imidophosphate, and p-nitrophenylphosphate did not substitute for ATP. The concentration of ATP required for half-maximal stimulation of Ca2+ uptake into the reconstituted vesicles was 6.2 microM. Magnesium was required for calcium uptake. Inhibitors of mitochondrial calcium-sequestering activities, i.e. oligomycin, sodium azide, ruthenium red, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and valinomycin did not affect the uptake of Ca2+ into the vesicles. In addition, strophanthidin and p-chloromercuribenzoate did not affect the transport. Calcium transport, however, was inhibited by vanadate in a concentration-dependent fashion with a K0.5 of 10 microM. A calcium-stimulated, vanadate-inhibitable phosphoprotein was demonstrated in the reconstituted vesicles with an apparent molecular weight of 118,000 +/- 1,300. These properties of Ca2+ transport by vesicles reconstituted from liver plasma membranes suggest that this ATP-dependent Ca2+ transport component is different from the high affinity (Ca2+-Mg2+)-ATPase found in the same membrane preparation (Lotersztajn, S., Hanoune, J. and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215; Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020). When the entire reconstituted vesicle population was treated with ATP and 45Ca in a buffer containing oxalate, the vesicles with Ca2+ transport activity could be separated from other vesicles by centrifugation in a density gradient and the ATP-dependent Ca2+ transport component was purified approximately 9-fold. This indicates that transport-specific fractionation may be used to isolate the ATP-dependent Ca2+ transport component from liver plasma membrane.
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PMID:Novel ATP-dependent calcium transport component from rat liver plasma membranes. The transporter and the previously reported (Ca2+-Mg2+)-ATPase are different proteins. 240 77

The characteristics of the H+ pump in isolated rat renal endocytotic vesicles were studied by the delta pH-sensitive dye acridine orange, the voltage-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide, and by a coupled optical ATPase assay. Intravesicular acidification depended on ATP and Mg2+ concentrations with half-maximal activations at 73 and 77 microM, respectively. CTP, GTP, UTP, and ITP partially supported acidification, but ADP and AMP did not. Ouabain, ethoxzolamide, levamisole, and vanadate did not inhibit H+ uptake into endocytotic vesicles. Oligomycin inhibited partially. Depending on concentration and preincubation time, Dio-9, filipin, N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD) inhibited H+ uptake completely. Filipin and, partially, DCCD acted nonspecifically by dissipating pH gradients. A specific cation was not required for the H+ pump; Zn2+ inhibited. Compared with mannitol, ATP-driven H+ uptake was stimulated by SCN- greater than Cl- greater than Br- greater than I- much greater than HPO4(2-) = gluconate = HCO3- = F-, but not by SO4(2-), NO3-, CH3COO-, S2O3(2-), and S4O6(2-). Chloride stimulated H+ uptake from the outside of the vesicles with an apparent Km of 27 mM. In the absence of Cl-, ATP-driven proton uptake was increased by intravesicular K+ and valinomycin, suggesting that the pump is electrogenic. The electrogenicity, however, could not be demonstrated with voltage-sensitive dyes. The vesicle membrane contains no significant K+ and Cl- conductances; only a conductance for H+ was found. The vesicles exhibited an ouabain-, oligomycin-, and vanadate-insensitive ATPase activity that was inhibited by DCCD and NEM. Our data indicate the presence of an electrogenic H+ pump in endocytotic vesicles from rat renal proximal tubules with similar characteristics as H+ pumps present in various intracellular (nonmitochondrial) membranes.
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PMID:Characteristics of the proton pump in rat renal cortical endocytotic vesicles. 242 58

We have characterized divalent-cation-stimulated nucleoside triphosphate hydrolase activity of the excitable ciliary membrane and compared it with a soluble Ca2+-ATPase released upon deciliation of Paramecium. The membrane-bound activity is strongly dependent on a divalent cation; calcium stimulates the basal activity of this enzyme at least 10-fold; magnesium and manganese stimulate less well, and strontium and barium, although less effective, also give measurable stimulation. This membrane-bound activity prefers ATP and GTP as substrates but also hydrolyzes UTP and CTP at measurable rates. The maximum velocity at saturating ATP concentrations and optimal calcium concentrations is 0.3 mumol/min per mg. The pH optimum for the membrane-bound activity is broad and centers around pH 7. From the temperature dependence of ATP hydrolysis, we calculate activation energies of 14 and 11 kcal/mol for the Ca2+- and Mg2+-stimulated activities, respectively. The Arrhenius plot is linear over the temperature range of 4 to 25 degrees C. The membrane ATPase is relatively insensitive to ouabain, oligomycin, N,N'-dicyclohexylcarbodiimide, vanadate, Ruthenium red and two calmodulin antagonists. Polyclonal antisera raised against the purified soluble ATPase from the deciliation supernatant show low reactivity with the membrane-bound ATPase. We conclude from the comparison of properties of the two activities that the ciliary membrane-bound ATPase is distinct from the soluble ATPase released by deciliation.
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PMID:Characterization of Ca2+- or Mg2+-ATPase of the excitable ciliary membrane from Paramecium tetraurelia: comparison with a soluble Ca2+-dependent ATPase. 242 1

The ability of ATP, CTP, ITP, GTP, UTP and two synthetic ATP analogs to provide for ouabain-sensitive Na+ accumulation into proteoliposomes with a reconstituted Na+,K+-ATPase (ATP phosphohydrolase, EC 3.6.1.37) was investigated. A correlation between the proton-accepting properties of the nucleotides and their ability to provide for active transport was found. The proton-accepting properties of the substrate seem to be a necessary condition for the shift from the K-form of Na+,K+-ATPase--an immutable step in the active translocation of Na+ and K+ through the Na+ pump.
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PMID:Na+ transport by reconstituted Na+,K+-ATPase in the presence of various nucleotides. 243 47


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