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

Abstract

ABSTRACr Microsomal vesicles of oat roots (Avena sativa var Lang) were separated with a linear dextran (0.5-10%, w/w) or sucrose (2545%, w/w) gradient to determine the types and membrne 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 C-and inhibited by NO-or 4,4'-diisothio-cyano-2,2'-stilbene disulfonic acid (DIDS). The distribution of C1-stimulated ATPase activity in a linear dextran gradient correlated with the distribution of H' pumping into vesicles as monitored by ['4Clmethyla-mine accumulation. The vanadate-inhibited ATPase was mostly insensitive to anions or DIDS and stimulated by K. These results show that microsomal vesices of plant tissues have at least two types of electro-genic, proton-pumping ATPases. The vanadate-insensitive and C1-stimulated , Hf-pumping ATPase may be enriched in vacuolar-type membranes ; the HW-pumping ATPase that is stimulated by K' and inhibited by vanadate is most likely associated with plasma membrane-type vesi-cles. The primary active transport process in higher plants is thought to be an electrogenic transport of protons (21, 25). The proton motive force generated by proton pumping can provide the driving force for transport ofcations, anions, amino acids, sugars, and hormones according to Mitchell's chemiosmotic hypothesis (19). Electrophysiological studies suggested that electrogenic proton pumps were localized in the plasma membrane (H+ extru-sion) (21, 25) and probably the tonoplast (H+ uptake) (16). The importance of such proton pumps to solute transport, as well as the regulation of growth and development, has made the study ' of proton transport an exciting research topic. Until recently, there was no direct evidence for a membrane constituent that pumped H+. Within the last few years, evidence for electrogenic, H+-pumping ATPases has appeared from our laboratory (4-6, 27-31) and several other laboratories (1, 7, 8, 10, 17, 18, 23, 24, 26, 32). H+-pumping ATPases have been identified in nonmitochon-drial membranes of several plant tissues. These transport ATP-ases exhibit three types ofactivities in sealed microsomal vesicles: (a) ionophore-stimulated ATPase activity (24, 27, 28), (b) ATP-dependent generation of a membrane potential (positive inside the vesicle) (1, 23, 26, 30), and (c) ATP-dependent pH gradient formation (acid inside) (1, 6, 8, 10, 18, 26, 29, 31, 32). Although several laboratories have demonstrated a vanadate-resistant, H+-pumping ATPase (8, 10, 18, 32), one report using oat roots (26) and our studies with sealed microsomal vesicles from tobacco callus or oat roots have suggested the presence of at least two types of H+-pumping ATPases, one sensitive and one insensitive to vanadate (5, 6, 28, 31). This paper demonstrates partial separation of two types of electrogenic, H+-pumping ATPases using a linear dextran or sucrose gradient. The two types of ATPases can be distinguished by their relative densities, K+ or Cl-sensitivities and sensitivity to inhibitors. The Cl-stimulated, proton pump appears to be enriched in vacuolar membranes and the K+-sensitive, proton pump is enriched in plasma membrane-type vesicles. Preliminary reports of these results have been presented (5, 31). MATERIALS AND METHODS Plant Material. Oat (Avena sativa L. var Lang) seedlings were germinated in the dark over an aerated solution of 0.5 mm CaSO4. After 5 to 6 days of growth, the apical tips (3-4 cm) of the roots were harvested. Lang oats were generously provided by the Agronomy Department of Kansas State University. Isolation and Separation of Sealed Microsomal Vesicles. Sealed microsomal vesicles were prepared as described by Churchill and Sze (6) using a 6% (w/w), 10%, or 12% dextran cushion. In one case, a two-step dextran gradient of 6% and 15% was used to separate vesicles at the 0/6% and 6/15% dextran interfaces. Microsomal vesicles (60,000g pellet) were sometimes separated with a linear dextran gradient (0.5-10%) as described before (6). Dextran rather than sucrose was chosen for most experiments as we found sucrose gradients did not improve membrane separation. Furthermore, we found slightly higher activities of pH gradient and membrane potential generation in vesicles prepared with dextran than with sucrose. Many of the dextran gradient tubes were intentionally overloaded with microsomal vesicles to permit membrane potential, pH gradient, and ATPase assays from the same gradient tube. Furthermore, vesicles were centrifuged for 2 h, and not to equilibrium, because vesicles often 921