The function of the Mge+-requiring KCI-stimuIated ATPase (ATP phosphohydrolase, EC 3.6.1.3) ofhigher plants in active ion transport was investigated by using a purified microsomal fraction containing sealed plasma membrane vesicles. Proc. NatL AcaS Sci USA 77, [5904][5905][5906][5907][5908]. A transmembrane electrical potential (+30 to +44 mV), monitored by uptake of a permeant anion (3SCN-), was generated specifically by ATP in Purified microsomal vesicles of tobacco callus. ATPdependent JaSCN-uptake required Mg2+, was optimal at pH 6.75, and showed similar ATP concentration dependence as the Mg2+-requiring KCI-stimulated ATPase activity. Plasma membrane ATPase inhibitors (N,N'-dicyclohexylcarbodiimide and vanadate) inhibited generation of the ATP-dependent electrical potential. A proton conductor (carbonyl cyanide m-chlorophenyihydrazone), but not a K+ ionophore (valinomycin), completely collapsed the electrical potential. The results provide in vitro evidence that the Mg24/KCI-ATPase of higher plants is an electrogenic pump. These results are consistent with the hypothesis that an electrogenic H' pump is catalyzed by the plasma membrane ATPase of plants.In higher plants, the primary active transport process is believed to be an electrogenic H+ pump (1, 2). The proton motive force generated could provide the driving force for transport of cations, anions, amino acids, and sugars, according to Mitchell's chemiosmotic principle (3). This H+ pump may be important in auxin-enhanced cell elongation, as the initial response ofcells to auxins is a stimulation of H+ secretion (4, 5). Several workers have postulated that the H+ pump is dependent on the hydrolysis ofATP catalyzed by a plasma membrane ATPase (ATP phosphohydrolase, EC 3.6.1.3) (6, 7). This hypothesis is supported by correlations observed between ATPase activities in vitro and changes of H+ fluxes and membrane potentials in intact cells (8)(9)(10)(11)(12)(13) MATERIALS AND METHODSPreparation of Sealed Microsomal Vesicles. Sealed microsomal vesicles were prepared by flotation of a microsomal fraction over a dextran pad, as described, by using autonomous tobacco callus, Nicotiana tabacum Linnaeus cv. Wisconsin no. 38 (16). These vesicles were relatively free of mitochondria, as judged by the low activity of mitochondrial enzymes (cytochrome c oxidase and malate dehydrogenase). Therefore, this fraction was referred to as purified microsomal vesicles. Protein was measured by the Lowry method using bovine serum albumin as the standard (17).ATPase Assay. ATPase activity was measured as described (16, 18) KCl-stimulated ATPase activity (KCl-ATPase) was determined as the difference in activity between Mg2+/KCl-ATPase and Mg2+-ATPase. Neither Mg2+-ATPase nor KCl-ATPase activity was affected by the presence of 1 mM KSCN (data not shown).35SCN-Uptake. mSCN-uptake was measured by a Millipore filtration method (19). SCN-uptake was initiated by adding an aliquot ofpurified microsomal vesicles to a reaction mixture. The final reaction mixture contained 25 mM ...

Subsequently, Sze and Churchill (32) reported that ATP generated a transmembrane potential (positive inside) and a pH gradient (acid inside) in sealed microsomal vesicles of tobacco callus and oat roots, respectively. Because the membrane potential was partially vanadate-sensitive, we concluded that a KCl-stimulated, Mg2+-requiring ATPase of the plasma membrane (33) is an electrogenic pump, but did not eliminate the possibility of electrogenic ATPases on other subcellular membranes. Recently, several laboratories have reported in vitro evidence of a H+-pumping ATPase from microsomal vesicles with properties different from a plasma membrane ATPase (5,8,10,21).In this paper, we show that there are at least two types of H+-pumping ATPases in nonmitochondrial membranes of plants: one is vanadate-sensitive and one is vanadate-insensitive. Proton pumping as measured by methylamine distribution is stimulated by Cl-and is insensitive to vanadate. MATERIALS AND METHODS Plant Material. Oats (Avena sativa L. var Lang) were germinated in the dark over an aerated solution of 0.5 mM CaSO4. After 5 to 6 d of growth, the apical tips (3-4 cm) of the roots were harvested. Lang oats were generously provided by the Agronomy Department, Kansas State University.Isolation of Sealed Microsomal Vesicles. The procedure of Sze (30) was used to isolate sealed membrane vesicles with some modifications. All procedures were conducted at 40C. Oat roots, usually 30 g, were homogenized twice with a mortar and pestle in a medium containing 250 mm mannitol, 3 mm EGTA2, 25 mm Hepes-BTP (pH 7.4), 1 mm DTT, 1 mM phenylmethylsulfonyl fluoride, and 0.5% BSA using a medium to tissue ratio of 3 ml/g. The brei was strained through cheesecloth and the homogenate was centrifuged for 15 min at 13,000g. The 13,000g supernatant was centrifuged for 30 min at 60,000g. The 60,000g pellet (crude microsomal fraction) was gently resuspended in a resuspension medium containing 250 mm mannitol, 2.5 mm Hepes-BTP (pH 7.3), and 1 mm DTT, layered onto a 6% (w/w) dextran cushion made in the resuspension medium (dextran average mol wt was 79,000), and centrifuged for 2 h at 70,000g. The white interface on top of the dextran cushion was collected and is referred to as the sealed microsomal vesicles. Protein concentration was estimated after precipitation with 10% TCA by the Lowry method (17), with BSA as the standard.

ABSTRACrMicrosomal 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 ( 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 nonmitochondrial membranes of several plant tissues. These transport ATPases exhibit three types ofactivities in sealed microsomal vesicles: (a) ionophore-stimulated ATPase activity (24, 27, 28), (b) ATPdependent 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 vanadateresistant, 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).

To underst the mecai and moleclr properties of the tonoplast-type HW-transocating ATPase, we have studied the effect of Cr, NOMj and 4,4'-dilsothocyano-2,2'-stilbe uf acid (DIDS) on the activity of the electrogenic H-ATPase associated with low-denity m icrosomal vehicles from oat roots (kenaa saia cv Lang). The We have distinguished two types of electrogenic, H+-pumping ATPases in nonmitochondrial membrane vesicles from oat roots (9, 39) and tobacco callus (35,36). One type is vanadate-insensitive and enriched in low-density membranes, such as the tonoplas, and one type is vanadate-inhibited and enriched in the plasma membrane fraction (9,39,40). Several other laboratories initially could demonstrate only a vanadate-resistant protonpumping ATPase in microsomal vesicles (2,14,16,24,25,30 (2,3,11,14,16,25,27,30,32,33). Our preliminary studies also showed that DIDS3 preferentially inhibited the anion-sensitive ATPase (9).To understand the mechanism and molecular properties of the vanadate-insensitive HW-pumping ATPase, we have examined in more detail the effect of Cl-, nitrate, and DIDS on three activities: (a) ATPase activity in the presence and absence of gramicidin; (b) ATP-generated membrane potential; and (c) ATP-dependent pH gradient formation. We concluded previously that the vanadate-insensitive ATPase, ApH, and Af activities are manifestations of an electronic Ht-pumping ATPase based on the parallel distributions of these activities on a linear dextran gradient (9). The present results ugest that the H+-ATPase has anion-sensitive site(s) which regulate its catalytic and vectorial activities. Preliminary results of this study have been reported (10, 39). MATERIALS AND MEFHODSPlant Material. Oat (Avena saliva L. var Lang) seedlings were germinated in the dark over an aerated solution of 0.5 mM CaS04. After 5 to 6 d of growth, the apical tips (3-4 cm) of the roots were harvested. Lang oats were generously provided by the Agronomy Department of the University of Maryland (College Park) and Kansas State University (Manhattan).Preparation of Sealed Microsonal Vesiles. Sealed microsomal vesicles were prepared as described by Churchill and Sze (11) using a 6% dextran (mol wt 69,000, Sigma) cushion. Vesicles containing the vanadate-insensitive ATPase are enriched in the 0/6% interface (9).Determination of ATP-GkemDrad ApH and As. A relative measure of the pH gradient (ApH) generated by inward H+ pumpinginto vesicles was determined as ['4Cqmethylamine (New England Nuclear) accumulation using a filtration procedure (1 1).Membrane potential (C& generation (inside positive) was determined as ATP-dependent SCN ('4C from Amersham or 3IS fiom New England Nuclear) uptake into vesicles using a filtration procedure described previously (38). We

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