Nigericin-stimulated ATPase activity in microsomal vesicles of tobacco callus

Abstract

The effect of ionophores on K(+)-stimulated adenosinetriphosphatase (ATPase; ATP phosphohydrolase, EC 3.6.1.3) activity of microsomal vesicles from tobacco callus was investigated. A nigericin-stimulated K(+)-ATPase activity was enriched in a purified microsomal fraction, which was obtained from the interphase of a dextran density gradient between 1.03 and 1.06 g/ml. The purified microsomal fraction was free of mitochondrial membranes and was composed partly of tightly sealed vesicles as indicated by the low K(+) permeability coefficient. The K(+)-dependent ATPase of this fraction was stimulated slightly by either carbonyl cyanide m-chlorophenylhydrazone (CCCP) (29%) or valinomycin (31%) alone; this ATPase was significantly stimulated by a combination of CCCP and valinomycin (73%) or by nigericin alone (80%). The K(+)-ATPase activity was stimulated by nigericin at pH 6.5 but not at pH 8.5. At pH 6.5, the K(+)-ATPase was inhibited by N,N'-dicyclohexylcarbodiimide but not by oligomycin. Nigericin stimulated the ATPase activity in the absence of initial KCl or pH gradients across the vesicle membrane. These results suggest that nigericin stimulates the ATPase activity by dissipating the H(+) or K(+) gradient or both, and support the hypothesis that the K(+)-ATPase mediates a H(+)/K(+) transport.