Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots

Abstract

When assayed in the presence of azide, NO(3) (-) was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 x M017) root microsomal membranes. The distribution of the NO(3) (-)-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H(+)-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 mum vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO(3) (-)-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H(+) from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K(+) > NH(4) (+) > Rb(+) > Cs(+) > Li(+) > Na(+) > choline(+)) whereas the NO(3) (-)-sensitive ATPase was stimulated by anions (Cl(-) > Br(-) > C(2)H(3)O(2) (-) > SO(4) (2-) > I(-) > HCO(3) (-) > SCN(-)). Both ATPases required divalent cations. However, the order of preference for the NO(3) (-)-sensitive ATPase (Mn(2+) > Mg(2+) > Co(2+) > Ca(2+) > Zn(2+)) differed from that of the vanadate-sensitive ATPase (Co(2+) > Mg(2+) > Mn(2+) > Zn(2+) > Ca(2+)). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO(3) (-)-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO(3) (-)-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO(3) (-)-sensitive ATPase was stimulated by gramicidin and was associated with NO(3) (-)-inhibitable H(+) transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO(3) (-)-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO(3) (-) sensitivity is a better criterion by which to identify this ATPase than either Cl(-) stimulation or H(+) transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.