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)

A variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of Neurospora crassa wild type strain 1A. A combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. Examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can only be removed upon prolonged incubation in the enzyme mixture (about 10 h). The 10-h incubation in the enzyme mixture appears to have no obvious detrimental effects on the integrity of the plasma membrane since the activity and regulatory properties of the glucose active transport system in 10-h spheroplasts are essentially unimpaired. Importantly, plasma membranes can be isolated from the 10-h spheroplasts by an adaptation of the concanavalin A method developed previously in this laboratory for cells of the cell wall-less sl strain, which is not the case for the 1.5-h spheroplasts. The yield of plasma membrane vesicles isolated by this procedure is 18-36% as indicated by surface labeling with diazotized [125I]iodosulfanilic acid, and the preparation is less than 1% contaminated with mitochondrial protein. The chemical composition of the wild type plasma membranes is similar to that previously reported for membranes of the sl strain of Neurospora. The isolated wild type plasma membrane vesicles also exhibit all of the functional properties that have previously been demonstrated for the sl plasma membrane vesicles. The wild type vesicles catalyze MgATP-dependent electrogenic proton translocation as indicated by the concentrative uptake of [14C]SCN- and [14C]imidazole under the appropriate conditions, which indicates that they contain the plasma membrane H+-ATPase previously shown to exist in the sl plasma membranes and that they possess permeability barrier function as well. The vesicles also contain a Ca2+/H+ antiporter as evidenced by their ability to catalyze protonophore-inhibited MgATP-dependent 45Ca2+ accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the isolated vesicles indicate that the protein composition of the wild type vesicles is roughly similar to that of the sl plasma membranes with the H+-ATPase present as a major band of Mr approximately 105,000. The wild type plasma membrane ATPase forms a phosphorylated intermediate similar to that of the sl ATPase, and the specific activity of the H+-ATPase in both wild type and sl membranes is approximately 3 mumol of Pi released/mg of protein/min.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Isolation and characterization of plasma membranes from strains of Neurospora crassa with wild type morphology. 622 20

Fluorescent probes have been used to measure electrogenic proton pumping by the plasma membrane ATPase of Neurospora. In isolated plasma membrane vesicles, greater than 85% of which are inverted, ATP hydrolysis is accompanied by the formation of an inside acid pH gradient (delta pH) which can be detected by acridine orange fluorescence quenching and an inside positive membrane potential (delta psi) which can be detected by oxonol V fluorescence quenching. Maximal values of delta pH were generated in the presence of a permeant anion (SCN-, NO-3, or Cl-) and maximal delta psi, in the absence of such anions. Cation effects were much less pronounced and can probably be accounted for by non-specific salt effects on the rate of ATP hydrolysis. In addition, a rapid method is described for the reconstitution of the [H+]-ATPase, starting from isolated plasma membranes. When the membranes are solubilized with deoxycholate in the presence of asolectin and detergent is removed by passage through a Bio-Gel P-10 column, vesicles are reformed in which the Mr = 104,000 polypeptide of the ATPase constitutes 35% of the protein. Freeze-fracture electron microscopy of the vesicles has revealed intramembrane particles with a diameter of 116 A, equally distributed between the two fracture faces. Measurements with acridine orange and oxonol V indicate that the reconstituted ATPase retains its transport activity, generating both delta pH and delta psi during the hydrolysis of MgATP.
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PMID:Electrogenic H+ translocation by the plasma membrane ATPase of Neurospora. Studies on plasma membrane vesicles and reconstituted enzyme. 623 6

The gills of both seawater and freshwater adapted eels have an ATPase activity which is stimulated by anions in the presence of Mg2+. Plasma membranes were distinguished from mitochondrial membranes with specific enzyme markers, the membrane fractions separated on a discontinuous sucrose gradient, and the ATPase activity of the plasma membranes studied. Activation by the anions of Cl- or HCO3- followed Michaelis-Menten kinetics and was competitively inhibited by SCN-. The Cl- and HCO3- activation characteristics were determined: no differences between the plasma membrane ATPase activities of freshwater and seawater-adapted fishes were observed. Maximal activity measurements after solubilization of the enzymes by Triton X 100 confirmed these findings. The function of a membrane anion-dependent ATPase in the brachial epithelium of euryhaline fish is discussed.
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PMID:Cl- -HCO3- dependent ATPase in gills of freshwater and seawater adapted eels (Anguilla anguilla L.). 626 Feb 35

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.
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PMID:Membrane potential of Plasmodium-infected erythrocytes. 628 30

A method is described for isolating plasma membrane vesicles from bovine tracheal epithelium. The procedure yields highly purified apical membranes which are enriched 19-fold in the marker enzyme, alkaline phosphatase. Contamination of this fraction by other organelles is minimal. Basolateral membranes isolated from the same preparation have a 4-fold enrichment of (Na+ + K+)-ATPase and a 2-fold reduction in alkaline phosphatase specific activity compared to the starting material. Assays of Na+ uptake by the apical membrane vesicles demonstrate their suitability for transport studies. Transport of Na+ into an intravesicular space was demonstrated by (1) a linear inverse correlation between Na+ uptake and medium osmolarity; (2) complete release of accumulated Na+ by treatment with detergent; and (3) a marked temperature-dependence of Na+ uptake rate. Other features of Na+ transport were (1) inhibition by amiloride; (2) insensitivity to furosemide; and (3) anion-dependence of uptake rate with the following selectivity:SCN- greater than Cl- greater than gluconate-.
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PMID:Isolation of transporting plasma membrane vesicles from bovine tracheal epithelium. 630 20

Membrane vesicles were purified from resting corpus mucosa of pig stomachs by velocity-sedimentation on a sucrose-Ficoll step gradient. Two vesicular fractions containing the (H+ + K+)-ATPase were obtained. One fraction was tight towards KCl, the other was leaky. At 21 degrees C maximal (H+ + K+)-ATPase activities of 0.8 and 0.4 mumol X mg-1 X min-1, respectively, were observed in lyophilized vesicles. The vesicles contained a membrane-associated carbonic anhydrase, the activity of which was in 100-fold excess of the maximal ATPase activity. Both vesicular fractions were rich in phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and cholesterol. The characteristics of ion permeability and transport in the tight vesicles were in agreement with corresponding data for vesicles of a tubulovesicular origin in the parietal cell. Measurement of the rate of K+ uptake into the vesicles was based on the ability of K+ to promote H+ transport. The uptake was slow and dependent on the type of anion present. The effectiveness in promoting uptake of K+ by anions was SCN- greater than NO3- greater than Cl- much greater than HCO3- greater than SO4(2-). Uptake of K+ was much more rapid at alkaline pH than at neutral or at acidic pH. Addition of CO2 at alkaline pH strongly stimulated the rate of H+ accumulation in the vesicles. The initial part of this stimulation was sensitive to acetazolamide, an inhibitor of carbonic anhydrase. A model how the (H+ + K+)-ATPase and the carbonic anhydrase may co-operate is presented. It is concluded that membrane vesicles of a tubulovesicular origin can produce acid.
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PMID:Characterization of proton-transporting membranes from resting pig gastric mucosa. 631 22

Hydrogencarbonate and chloride activated, ouabain-insensitive ATPase activities are demonstrated in the salt-absorbing rectum of larval dragonflies. Maximal activation is achieved at approx. 30 mM HCO3- and 20 mM Cl-, respectively. The stimulation of each anion obeys Michaelis-Menten kinetics Km values are 4.65 mM for HCO3-- and 10.25 mM for Cl--activation. The activating anion of one type of ATPase simultaneously exerts an inhibitory effect on the other. Cl--activation is also reduced by Mg.ATP in concentrations above 0.5 mM and by Tris-Hepes buffer exceeding 2.5 mM. Both anion-dependent ATPase activities are found enriched in subcellular membraneous fractions of the rectum. Thiocyanate inhibits both activities and causes a significant decrease in rectal uptake of radioactive chloride from hypo-osmotic external solution. In the case of HCO3- dependent ATPase a competitive inhibition as SCN- was found with an inhibitor constant of Ki=0.5 mM.
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PMID:[Biochemical demonstration of HCO3--und Cl--dependent ATPase activities in the rectum of larval dragonflies and inhibition of rectal chloride uptake by thiocyanate (author's transl)]. 644 80

The intracellular localization of a Cl--HCO3--ATPase, which is inhibited by SCN-, was studied in the gills of the rainbow trout, Salmo gairdneri. This activity can be measured in the absence of contamination by mitochondria (i.e., in the absence of succinate dehydrogenase or cytochrome c oxidase activities). The distribution of the 5'-nucleotidase and of the ATPase stimulated by Cl- and HCO3- after sucrose density gradient centrifugation of the microsomal fraction was compared. Because those activities cannot be separated, it is postulated that the anion-stimulated ATPase is located in the plasma membrane. The activation of this microsomal anion ATPase by chloride has been studied extensively. The possible role of the Cl--HCO3--ATPase of trout gills in the Cl-/HCO3- exchange and in the regulation of the internal acid-base balance is discussed.
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PMID:Cl--HCO3--ATPase in gills of the rainbow trout: evidence for its microsomal localization. 644 66

The transmembrane electropotential of microsomal vesicles from pea internode segments, monitored by equilibrium distribution of the permeant anion SCN-, is strongly hyperpolarized when ATP is present in the incubation medium. The stimulation of SCN- uptake by ATP is rather specific with respect to the other nucleoside di- and triphosphates tested: ADP, GTP, CTP and UTP. ATP-stimulated SCN- uptake is strongly inhibited by ATPase inhibitors such as p-chloromercuribenzenesulphonate and N,N'-dicyclohexylcarbodiimide and by 2.5% toluene/ethanol (1 : 4, v/v), the latter being a treatment which makes the vesicles permeable. On the contrary, oligomycin is almost ineffective in influencing ATP-induced SCN- uptake. The proton conductor carbonyl cyanide p-trifluoromethoxyphenylhydrazone strongly inhibits ATP-stimulated SCN- uptake. The effect of ATP on SCN- uptake depends on the pH of the medium, the maximum being reached at about pH 7.0. These data support the view that microsomal fractions from pea internodes contain membrane vesicles endowed with a membrane-bound ATPase coupling ATP hydrolysis to electrogenic transport of ions, probably H+.
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PMID:Evidence for an electrogenic ATPase in microsomal vesicles from pea internodes. 645 6

When current was sent from serosa (S) to mucosa (M) across the frog stomach, there was a polarization (POL) of the open circuit potential (OCPD). POL was not affected by NaCl-free solutions, but was decreased by inhibitors of the H+ pump. In present experiments, current was sent to clamp the PD (VC) across the mucosa in steps of 20 mV up to 100 mV below the control OCPD, that is, current was sent from M to S. All experiments were performed in NaCl-free solutions. The POL was expressed as a % of the difference between the VC PD and the control OCPD. In 4 mM K+ control solutions, the POL was 11.8%; with 10(-3) M omeprazole (H+/K+ pump inhibitor), 1.1; with 10(-5) M SCH 28080 (H+/K+ pump inhibitor), 3.6; with 10(-3) M famotidine (H2 blocker), 1.6; and with 10(-2) M SCN-, 25.4 (inhibition of H+ sec, but not of the pump); in 79 mM K+ control solutions, 26.2; with 10(-3) M omeprazole, 4.2; with 10(-5) M SCH 28080, 15.9; with 10(-3) M famotidine, 5.6; and with 10(-2) M SCN-, 29.9. POL was higher in high K+ than in low K+ solutions contrary to what was observed in previous experiments with current sent from S to M. Results are explained on the basis of an electrogenic H+/K(+)-ATPase pump which includes a H+ channel, permeable to K+. With high K+ solutions, K+ is driven through the H+ channel onto the antiporter (ATPase) when current is sent from M to S, resulting in a greater POL of the pump.
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PMID:Effect of current direction and K+ on polarization of the frog gastric mucosa proton pump. 749 46


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