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)

ATPases were isolated from chloroplasts of the unicellular marine alga Acetabularia acetabulum. Two preparations of ATPase, a chloroplast-enriched fraction and an alpha beta gamma-complex were compared. The alpha beta gamma-complex was released into an EDTA solution and purified by anion-exchange chromatography, hydrophobic chromatography, and gel permeation chromatography. The subunit composition of this enzyme appeared to be 52-53 (alpha), 51 (beta), and 40 (gamma) kDa from SDS-PAGE. ATPase activity was enriched about 260-fold to a specific activity of approximate 4.1 U.mg protein-1. The catalytic properties of the alpha beta gamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (Km for ATP 0.2 mM); divalent cation requirement, Mg2+ = Mn2+ = Co2+ > Zn2+ > Ni2+ > Ca2+; ATPase activity was inhibited by monovalent anions (NO3-, SCN-), while monovalent cations had neither inhibitory nor stimulatory effect. Orthovanadate had no inhibitory effect on the enzyme activity of alpha beta gamma-complex. Azide was the most effective inhibitor of the alpha beta gamma-complex. N-Terminal amino acid sequences of the alpha and beta subunits were not obtained and appeared to be blocked. The gamma subunit gave a sequence of AGLKEMKD-XIGSVXNTKKI, which showed 60% similarity to the gamma subunits of spinach and Chlamydomonas reinhardtii CF1-ATPase and EF1-ATPase.
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PMID:Chloroplast ATPase in Acetabularia acetabulum: purification and characterization of chloroplast F1-ATPase. 776 91

ATP hydrolysis induces the activation of the proton ATPase in chromatophores of Rhodobacter capsulatus supplemented with nigericine and 50 mM K+ (i.e. when delta pH < 0.2 units). The value of transmembrane electric potential (delta phi) driving this activation was measured using three different approaches: carotenoid electrochromism, uptake of SCN- and responses of the dye oxonol VI. The value of delta phi calculated from the SCN- uptake, on the basis of an internal volume determined experimentally, was about 140 mV, while that indicated by the electrochromic signal ranged between 35 and 70 mV. Only the value indicated by SCN- distribution is consistent with the energetic requirement for the activation of H(+)-ATPase.
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PMID:Unreliability of carotenoid electrochromism for the measure of electrical potential differences induced by ATP hydrolysis in bacterial chromatophores. 779 13

The Ca(2+)-ATPase activity of the trypsin-activated CF1 presented a monophasic pattern, indicating that the active centres of the enzyme were acting with the same kinetic properties. The study of the effect of the anions cianate (OCN-) and thiocyanate (SCN-) on the ATPase activity showed the existence of cationic regulatory sites, capable of binding these modulators in a competitive way, resulting in the inhibition of the ATPase activity. Nucleotides ADP and ATP, at high concentrations, were competitive inhibitors for the substrate Ca(2+)-ATP. ATP, at low concentrations, presented an activating effect. The study of the combined effects of ATP (at low concentrations) and SCN- on ATPase activity revealed the existence of a non-competitive relationship between anions and nucleotides. The modification of CF1 with fluorescein isothiocyanate, a specific reagent that binds to amino groups of nucleotide binding centres, yielded a molar relationship FITC/CF1 = 4, both with the trypsin-treated and non treated enzyme. This specific incorporation took place on the alpha and, beta subunits of CF1, and resulted in a decrease of about 30% of the ATPase activity. These results are consistent with the existence of either three catalytic and three regulatory sites or four catalytic and two regulatory sites on CF1.
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PMID:Catalytic and regulatory sites in CF1. 799 41

Recently, we have shown that polarization of an electrogenic H+/K(+)-ATPase pump located in the secretory (luminal) membrane of the frog gastric mucosa is the major factor contributing to the increase in open circuit potential difference (OCPD) induced by voltage clamping. While this transmucosal polarization was not affected by removal of Cl- and Na+ and minimally affected by increasing the K+ concentration to 79 mM in both nutrient and secretory solutions, it was markedly reduced by 10(-3) M famotidine (beta blocker) or 10(-4) M omeprazole (H+/K(+)-ATPase inhibitor) in the nutrient solution. In present experiments, the effects of three other inhibitors of H+ secretion were examined, namely, cimetidine (beta blocker), SCH 28,080 (H+/K(+)-ATPase inhibitor) and SCN- (non-specific inhibitor). While cimetidine and SCH 28,080 markedly reduced the polarization induced by voltage clamp, SCN- affected the polarization to a lesser extent. These data further support the electrogenicity of the frog gastric mucosa proton pump and the lack of a direct effect of SCN- on the pump.
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PMID:Electrogenicity of the frog gastric mucosa proton pump based on polarization responses in the presence of H(+)-secretion inhibitors. 832 38

The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.
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PMID:H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes. 838 68

CFTR shares structural homology with the ABC transporter superfamily of proteins which hydrolyze ATP to effect the transport of compounds across cell membranes. Some superfamily members are characterized as P-type ATPases because ATP-dependent transport is sensitive to the presence of vanadate. It has been widely postulated that CFTR hydrolyzes ATP to gate its chloride channel. However, direct evidence of CFTR hydrolytic activity in channel gating is lacking and existing circumstantial evidence is contradictory. Therefore, we evaluated CFTR chloride channel activity under conditions known to inhibit the activity of ATPases; i.e., in the absence of divalent cations and in the presence of a variety of ATPase inhibitors. Removal of the cytosolic cofactor, Mg2+, reduced both the opening and closing rates of CFTR suggesting that Mg2+ plays a modulatory role in channel gating. However, channels continued to both open and close showing that Mg2+ is not an absolute requirement for channel activity. The nonselective P-type ATPase inhibitor, vanadate, did not alter the gating of CFTR when used at concentrations which completely inhibit the activity of other ABC transporters (1 mM). Higher concentrations of vanadate (10 mM) blocked the closing of CFTR, but did not affect the opening of the channel. As expected, more selective P-type (Sch28080, ouabain), V-type (bafilomycin A1, SCN-) and F-type (oligomycin) ATPase inhibitors did not affect either the opening or closing of CFTR. Thus, CFTR does not share a pharmacological inhibition profile with other ATPases and channel gating occurs in the apparent absence of hydrolysis, although with altered kinetics. Vanadate inhibition of channel closure might suggest that a hydrolytic step is involved although the requirement for a high concentration raises the possibility of previously uncharacterized effects of this compound. Most conservatively, the requirement for high concentrations of vanadate demonstrates that the binding site for this transition state analogue is considerably different than that of other ABC transporters.
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PMID:Lack of conventional ATPase properties in CFTR chloride channel gating. 866 89

The volume response of vestibular dark cells of the gerbil to a hyposmotic challenge was investigated. Tissues including dark cells were perfused in preparations in which the perfusate had access to both sides of the epithelium and the height of the dark cell layer was measured as an indicator of its volume. We found that dark cells showed a fast and strong regulatory volume decrease (RVD) and prevented cell swelling in hypotonic media. This mechanism was dependent upon extracellular [K+] and [Cl-]. Ion selectivity of this mechanism was K+ = Rb+ > Cs+ > Na+ = NMDG+ (N-methyl-D-glucamine) for cations and Cl- = SCN- = NO3- > > gluconate- for anions. RVD of dark cells was inhibited by K(+)- channel blockers barium, quinidine and lidocaine, by Cl(-)-channel blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, by an Na(+)-K+ ATPase inhibitor ouabain and by low temperature, but was not inhibited by a loop diuretic bumetanide, by carbonic anhydrase inhibitors acetazolamide and ethoxyzolamide, by a K(+)-channel blocker tetraethylammonium, by a Cl(-)-channel blocker 5-nitro-2 (3-phenylpropylamino)-benzoic acid or by an inhibitor of the Na(+)-H+ exchanger amiloride. These data suggest that the RVD of dark cells occurs via separate K+ and Cl- channels which are different from those active under isosmotic condition, and is presumably activated by a hyposmotic stimulus.
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PMID:[Mechanism of volume regulation of vestibular dark cells of the gerbil]. 875 75

We have shown that polarization of an electrogenic H+/K+ ATPase pump located in the secretory (luminal) membrane of the frog gastric mucosa is the major factor contributing to the change in open circuit potential difference (OCPD) induced by voltage clamping. This transmucosal polarization was markedly reduced by H2 blockers famotidine and cimetidine, and by the H+/K+-ATPase inhibitors omeprazole and SCH 28080. SCN-, a nonspecific H+ secretion inhibitor, did not affect the polarization. In the present experiments, the effects of two other inhibitors of H+ secretion were examined, namely, acetazolamide (AA), a carbonic anhydrase inhibitor, and melittin (MEL), an inhibitor of the H+/K+-ATPase enzyme. When AA 10(-3) M or MEL 10(-5) M was added to the nutrient solution, H+ secretion was completely inhibited. While MEL markedly reduced the polarization induced by voltage clamp, AA did not affect the polarization. These data support the concept that MEL directly affects the electrogenic H+/K+-ATPase pump while the inhibition of H+ secretion by AA is by an indirect mechanism. The data further support the electrogenicity of the H+/K+-ATPase.
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PMID:Effect of acetazolamide and melittin on polarization of the frog gastric mucosa proton pump. 898 9

It was found that a facultatively anaerobic and halophilic alkaliphile, M-12 (Amphibacillus sp.), possesses a Na(+)-stimulated ATPase in the membrane. The ATPase activity was inhibited by NO3- and SCN- which are the inhibitors of V-type ATPase, but not by azide and vanadate, inhibitors of F-type ATPase and P-type ATPase, respectively. Upon the incubation of the membrane in buffer containing ATP and MgCl2, several polypeptides were released from the membrane. Among them, two major polypeptides with apparent molecular masses of 79 and 55 kDa crossreacted with an antiserum against the catalytic units (subunits A and B) of V-type ATPase from Enterococcus hirae. The N-terminal amino acid sequences of the 79 and 55 kDa polypeptides showed high similarity to those of subunits A and B of V-type ATPase from Enterococcus hirae, respectively. M-12 is likely to possess a V-type Na(+)-ATPase.
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PMID:Presence of Na(+)-stimulated V-type ATPase in the membrane of a facultatively anaerobic and halophilic alkaliphile. 978 52

The plasma-membrane potential (Delta(psi)p) in bloodstream forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN- were used to report Delta(psi)p directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Delta(psi)p only when Delta(psi)m had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Delta(psi)p had a value of -82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (-76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Delta(psi)p together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl- by the gluconate anion on Delta(psi)p. The contribution to Delta(psi)p by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Delta(psi)p.
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PMID:Factors that determine the plasma-membrane potential in bloodstream forms of Trypanosoma brucei. 1090 93


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