Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies.
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[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl 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.
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D009492:Neurospora crassa / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000032:analysis; D012440:Saccharomyces / Q000201:enzymology; D003313:Zea mays / Q000201:enzymology*
D046911:Macromolecular Substances; D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan