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 2SO 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.