Antimycin A inhibition of proton pumping associated with root plasma membrane H+-ATPase

Plasma membrane vesicles obtained by discontinuous sucrose gradient centrifugation from corn roots, exhibited vanadate-sensitive H+-ATPase activity with minimum contaminations of other subcellular ATPases and phosphatase activities. The H+-ATPase catalyzed the hydrolysis of Mg-ATP and supported a proton pumping process. The presence of antimycin A had no significant effects on the rate of catalyzed ATP hydrolysis. However, proton pumping activity was substantially reduced. Time courses of proton pumping were analyzed by a kinetic model which allowed an accurate determination of the initial pumping rate (R-0) and the proton leakage of the energized membrane (k(1)) from steady-state properties. With a rapid quenching of ATP hydrolysis by the use of hexokinase, the proton leakage of de-energized membrane (k(2)) was also determined This analysis indicated that antimycin A inhibition was characterized by a decrease in R-0 without any significant effects on both k(1) and k(2). Possible implications of this differential inhibition on coupled ATPase activities ape discussed.