Separation of Two Types of Electrogenic H+-Pumping ATPases from Oat Roots

Abstract

Microsomal vesicles of oat roots (A. sativa cv. Lang) were separated with a linear dextran (0.5-10%, wt/wt) or sucrose (25-45%, wt/wt) gradient to determine the types and membrane identity of proton-pumping ATPases associated with plant membranes. ATPase activity stimulated by the H+/K+ exchange ionophore nigericin exhibited 2 peaks of activity on a linear dextran gradient. ATPase activties or ATP-generated membrane potential (inside positive), monitored by SCN- [isothiocyanide] distribution, included a vanadate-insensitive and a vanadate-sensitive component. ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was partially inhibited by vanadate. The vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose); the vanadate-sensitive activity was enriched at 4-7% dextran or 32-37% sucrose. The low-density ATPase was stimulate by Cl- and inhibited by NO-3 or 4,4''-diisothiocyano-2,2''-stilbene disulfonic acid (DIDS). The distribution of Cl--stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H+ pumping into vesicles as monitored by [14C]methylamine accumulation. The vandate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K+. Microsomal vesicles of plant tissues have at least 2 types of electrogenic, proton-pumping ATPases. The vanadate-insensitive and Cl-stimulated, H+-pumping ATPase may be enriched in vacuolar-type membranes; the H+-pumping ATPase that is stimulated by K+ and inhibited by vanadate is most likely associated with plasma membrane-type vesicles.