Localization of a Proton-Translocating ATPase on Sucrose Gradients

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Abstract
Ionophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 cm of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome [Cyt] c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the 1st 4 cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase activity coincided in a broad band extending from 1.08-1.15 g per cm2 with maximum activity at 1.10-1.12 g/cm2. Large portions of the proton-translocating ATPase activity and ionophore-stimulated ATPase activity were clearly separable from mitochondrial membranes containing Cyt c oxidase activity and azide-sensitive, pH 8.5 ATPase activity and from membranes bearing .beta.-glucan synthetase I and II. The vesicles coincided with a minor portion of the NADH-Cyt c reductase and nucleoside diphosphatase activities. Evidently, the vesicles are of tonoplast origin.