title: H+-ATPase of crude homogenate of the outer mantle epithelium of Anodonta cygnea.
abstract: The outer mantle epithelium of the freshwater bivalve, Anodonta cygnea, is responsible for the mineralisation of the shell. Under short circuit conditions, it generates a current that is due to the operation of an electrogenic proton pump located in the apical barrier of that epithelium. Bafilomycin A1 and Concanamycin A inhibited the short circuit current. The IC50 and maximum inhibition dose were 0.17 and 0.5 microM for Bafilomycin A1, and 0.7 and 5 microM for Concanamycin A. The present work was undertaken to further characterise V-type ATPase of the outer mantle cells. The V-ATPase enzymatic activity of crude homogenate, measured as the amount of inorganic phosphorous released, due to ATP hydrolysis, in the presence of Na2SO3 (200 mM) was found to be 4.6+/-1.1 micromol Pi/mg protein/h, at 27 degrees C, pH 7.0-7.4 and ATP 4.5-6.0 microM. Bafilomycin A1 and Concanamycin A inhibit the V-ATPase activity with an IC50 of 14 and 8 nmol mg(-1), respectively. Dicyclohexylcarbodiimide (DCCD; 100 mM) and NaNO3 (100 microM) inhibited the V-type ATPase in what it seems a non-specific manner and NEM (100 mM) was unable to do it. Bafilomycin A1 (10 microM) and Concanamycin A (10 microM), inhibited 50-60% of the total activity.
authors.affiliation: Transporte Iónico e Fisiologia dos Gâmetas, CECA-UP, Porto, Portugal.
authors.forename: P F
authors.initials: PF
authors.lastname: Oliveira
authors.forename: I A
authors.initials: IA
authors.lastname: Lopes
authors.forename: C
authors.initials: C
authors.lastname: Barrias
authors.forename: A M
authors.initials: AM
authors.lastname: Rebelo da Costa
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D018942:Macrolides / Q000494:pharmacology; D008974:Mollusca / Q000033:anatomy & histology / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D009566:Nitrates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013696:Temperature
chemical_list: D002457:Cell Extracts; D004791:Enzyme Inhibitors; D018942:Macrolides; D009566:Nitrates; C036978:concanamycin A; C040929:bafilomycin A1; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases