Proton-translocating ATPase activity in plasma membrane vesicles from roots of grapevine seedlings

Abstract

Microsomal vesicles isolated from grape ( Vitis vinifera, cv. Verduzzo) roots were shown to possess ATP-dependent and vanadate-inhibited proton-translocating activity which was inhibited 30% by oligomycin and was insensitive to nitrate. Contamination by mitochondrial H +-ATPase activity was strongly reduced by modifying the extraction procedure and by collecting vesicles with density lower than 1.18 g × cc −1 after centrifugation of microsomes for 2 h at 95 000 × g on a 40% sucrose cushion. Proton transport was inhibited 65% by 200 μM vanadate. The half-maximal inhibition occured at 100 μM. Optimum of the proton transport activity pH was 6.5 and the Mg:ATP complex was strongly preferred substrate. Intravesicular acidification was dependent on the anion accompanying K + ions (Br − > Cl − > NO 3 − ⪢ SO 4 2− > IDA −) and it was stimulated by K + ions, as shown by addition of K-IDA after incubation of vesicles with BTP-Cl or BTP-NO 3. Enzymatic hydrolysis of Mg:ATP was stimulated 42% by K-IDA and inhibited 76% by vanadate. Oligomycin and nitrate showed almost no effect (16 and 5% inhibition, respectively). Results indicate that membrane vesicles isolated by this procedure from grape roots mainly derive from the plasma membrane representing the necessary material to study basal phenomena of mineral nutrition of grape plants. Agronomic and ecological adaptability and genetic variability make grape cultivars a good plant material to study factors controlling nutrition efficiency and regulation.