REINVESTIGATION OF AUXIN AND FUSICOCCIN STIMULATION OF THE PLASMA-MEMBRANE H-ATPASE ACTIVITY()

In-vivo treatment of maize (Zea mays L.) coleoptile segments with auxi n (indole-3-acetic acid; IAA) and fusicoccin (FC) followed by plasma-m embrane isolation was used to characterize the effects of these treatm ents on the plasma-membrane H+-ATPase. Both IAA and FC increased H+ ex trusion and elongation rate of the coleoptile segments, FC more strong ly than IAA. Plasma membranes isolated after in-vivo treatment with FC showed a twofold stimulation of ATP hydrolysis and a several-fold sti mulation of H+ pumping, whereas no effect was observed after IAA treat ment, irrespective of whether the plasma membranes were prepared by tw o-phase partitioning or sucrose-gradient centrifugation. A more detail ed investigation of the kinetic properties and pH dependence of the en zyme showed that FC treatment led to a twofold increase in V-max, a de crease in K-m for ATP from 1.5 mM to 0.24 mM, and a change in pH depen dence resulting in increased activity at physiological pH levels. Agai n, IAA treatment showed no effects. Quantitation of the H+-ATPase by i mmunostaining using four different antibodies revealed no difference b etween IAA-and FC-treated material, and controls. From these data we c onclude that (i) neither IAA nor FC gives rise to an increase in the a mount of H+-ATPase molecules in the plasma membrane that can be detect ed after membrane isolation, and (ii) if the H+-ATPase is activated by IAA, this activation is, in contrast to FC activation, not detectable after membrane isolation.