ATP‐dependent H⁺‐pumping of a low‐density fraction of pea stem microsomes

Abstract

A low‐density fraction of pea (Pisum sativum L. cv. Alaska) stem microsomes, obtained from a discontinuous sucrose gradient, possessed an H+‐ATPase able to generate a proton gradient and an electrical potential. The proton pumping was insensitive to monovalent cations, to vanadate and oligomycin, required a permeant anion and was inhibited by nitrate, N, N'‐dicyclohexylcarbodiimide and diethylstilbestrol. The H+‐ATPase had a pH optimum around 6.0–6.5 and was saturable with respect to the substrate Tris‐ATP (Km≅ 0.4 mM). Ca2+ (0.05–1 mM) induced a dissipation of the ATP‐generated δpH without affecting ATPase activity. At physiological concentrations (1–5 mM), nitrate caused an initial slight increase of the ATP‐generated proton gradient followed by a complete dissipation after 2–3 min. The dissipating effect was not caused by inhibition of ATPase activity, since ATP prevented the nitrate‐induced collapse of δpH. On the other hand, ATPase activity, evaluated as release of Pi, was not inhibited by concentrations lower than 20 mM KNO3. These results indicate that nitrate entered the vesicles in response to an electrical potential and then could exit in symport with protons, while Ca2+ entered in exchange for protons (antiport).