Anion-Sensitive, H⁺-Pumping ATPase in Membrane Vesicles from Oat Roots

Abstract

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.

ADP‐ or pyrophosphate‐dependent proton pumping of pea stem tonoplast‐enriched vesicles

“…Recently, the evidence for a PPiase in the generation of an electrochemical proton gradient across tonoplast, microsomal and Golgi membranes has been found [8][9][10][11][12].…”

Characterization of the Effects of Divalent Cations on the Coupled Activities of the H+-ATPase in Tonoplast Vesicles

“…The proton electrochemical potential, AUH+, which contains a ApH (acidic inside) and a A'I (positive inside) generated by these pumps serves as the driving force for the secondary transport of other solutes across tonoplast (3,16,17). The tonoplast H+-ATPase requires Mg-ATP as the substrate and can be inhibited by nitrate ions (7,19). The properties of this enzyme, e.g.…”

Differential Inhibition of Tonoplast H+-ATPase Activities by Fluorescamine and Its Derivatives

“…This discharge, in essence, represents the membrane proton leakage in the deenergized state. We demonstrated previously (29) that this process can be described by a simple first-order decay equation: I n(6/6,) = -k2t (8) in which 6 and k2 represent the residual proton gradient and the leakage constant of deenergized membrane, respectively.…”

“…The tonoplast membrane of plant roots contains at least two enzymes, an ATPase and a pyrophosphatase, which can convert the chemical free energy released from the hydrolysis of high-energy phosphoester bonds, into a transmembrane proton, electrochemical gradient (2,6,8,33). According to the chemiosmotic concept, the resulting pH gradient and membrane potential may be used as the primary driving force to transport materials across the membrane (15,19).…”

“…Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.The tonoplast membrane of plant roots contains at least two enzymes, an ATPase and a pyrophosphatase, which can convert the chemical free energy released from the hydrolysis of high-energy phosphoester bonds, into a transmembrane proton, electrochemical gradient (2,6,8,33). According to the chemiosmotic concept, the resulting pH gradient and membrane potential may be used as the primary driving force to transport materials across the membrane (15,19).…”