Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies (original) (raw)
1986, Journal of Biological Chemistry
Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72-and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro['4C]benzo-2-oxa-1,3-diazole (['4C]NBD-Cl) and N,N'-['4C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-C1 preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit. Plant vacuoles are acidic organelles in which ions, sugars, organic acids, and hydrolytic enzymes are stored (1). Studies with isolated vacuoles and tonoplast (plant vacuolar membrane) vesicles have indicated that a proton-translocating ATPase present on the tonoplast generates an electrochemical gradient, which may be responsible for the observed accumulation of ions and solutes (reviewed in Ref. 2). A similar ATP-dependent proton pump is also present on the Golgi of maize coleoptiles (3). Several recent reports have described the partial purification of a novel ATPase from plant and ~~
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