Characterization of <i>In Vitro</i> Proton Pumping by Microsomal Vesicles Isolated from Corn Coleoptiles

Abstract

Corn (Zea mays L. cv Golden Cross Bantam) coleoptile microsomal vesicles have been isolated which are capable of ATP-driven H(+)-transport as measured by [(14)C]methylamine accumulation and quinacrine fluorescence quenching. Formation of the pH gradient in vitro shows a high specificity for ATP.Mg, is temperature-sensitive, exhibits a pH optimum at 7.5, and is inhibited by carbonyl cyanide-m-chlorophenylhydrazone. Of the divalent cations tested, Mn(2+) is almost as effective as Mg(2+), while Ca(2+) is ineffective. Excess divalent cations, particularly Ca(2+), reduces the pH gradient. H(+) transport is strongly promoted by anions, especially chloride, while potassium does not affect pump activity. Studies with (36)Cl(-) indicate that ATP-driven H(+) transport into the vesicles is associated with chloride uptake. Both carbonyl cyanide-m-chlorophenylhydrazone and the anion transport inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, inhibit methylamine accumulation and (36)Cl(-) uptake. Proton pumping is also blocked by diethyl stilbestrol and N,N'-dicyclohexylcarbodiimide, but is insensitive to oligomycin and vanadate. These properties of the pump are inconsistent with either a mitochondrial or plasma membrane origin.