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 A 1 and Concanamycin A inhibited the short circuit current. The IC 50 and maximum inhibition dose were 0.17 and 0.5 μM for Bafilomycin A 1 , and 0.7 and 5 μM 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 Na 2 SO 3 (200 mM) was found to be 4.6±1.1 μmol Pi/mg protein/h, at 27 °C, pH 7.0–7.4 and ATP 4.5–6.0 μM. Bafilomycin A 1 and Concanamycin A inhibit the V-ATPase activity with an IC 50 of 14 and 8 nmol mg −1 , respectively. Dicyclohexylcarbodiimide (DCCD; 100 mM) and NaNO 3 (100 μM) inhibited the V-type ATPase in what it seems a non-specific manner and NEM (100 mM) was unable to do it. Bafilomycin A 1 (10 μM) and Concanamycin A (10 μM), inhibited 50–60% of the total activity.