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

Abstract

Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (25-45%, w/w) 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 two peaks of activity on a linear dextran gradient. ATPase activities or ATP-generated membrane potential (inside positive), monitored by SCN(-) distribution, included a vanadate-insensitive and a vanadate-sensitive component. In a previous communication, we reported that ATP-dependent pH gradient formation (acid inside), monitored by quinacrine fluorescence quenching, was also partially inhibited by vanadate (Churchill and Sze 1983 Plant Physiol 71: 610-617). Here we show that the vanadate-insensitive, electrogenic ATPase activity was enriched in the low density vesicles (1-4% dextran or 25-32% sucrose) while the vanadate-sensitive activity was enriched at 4% to 7% dextran or 32% to 37% sucrose. The low-density ATPase was stimulated 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 [(14)C]methylamine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K(+). These results show that microsomal vesicles of plant tissues have at least two 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.