Irreversible inhibition of H +-ATPase of higher plant tonoplast by chaotropic anions: evidence for peripheral location of nucleotide-binding subunits

Abstract

The H +-translocating ATPase of tonoplast vesicles from storage root of Beta vulgaris L. is irreversibly inhibited by a 30 min treatment with the chaotropic anions SCN −, ClO 4 −, I − or NO 3 − in the 0.25–1.5 M concentration range. The inhibitory potencies of the anions follow the Hofmeister series ( SCN − > ClO 4 − > I − > NO 3 − > CH 3 COO − ; SO 4 2− = 0 ). The H +-translocating inorganic pyrophosphatase of the same membrane is, by contrast, unaffected by chaotrope concentrations which completely abolish H +-ATPase activity. Inhibition of the ATPase is associated with the removal of two polypeptides of 67 and 57 kDa from the membrane, concomitant with their appearance in the supernatant. The chaotrope-dissociated 67 and 57 kDa polypeptides comigrate with the major subunits of the partially purified ATPase upon SDS-polyacrylamide gel electrophoresis and cross-react with antibody raised to the nucleotide-binding subunits of the enzyme. Since the 16 kDa [ 14C]DCCD-binding proteolipid of the ATPase remains associated with the membrane after treatment with chaotrope, it is concluded that chaotropic anions inhibit the enzyme by specific detachment of the nucleotide-binding subunits. The tonoplast ATPase of Beta is therefore deduced to have structure/function partitioning analogous to the F 0F 1 H +-ATPase of energy-coupling membranes.