PREPARATION OF MEMBRANE-VESICLES ENRICHED IN ATP-DEPENDENT PROTON TRANSPORT FROM SUSPENSION-CULTURES OF TOMATO CELLS

Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells. Suspension cultures were a source of large quantities of membranes from rapidly growing, undifferentiated cells. Proton transport activity was assayed as quench of acridine orange fluorescence. The activity of the proton translocating ATPase and of several other membrane enzymes was measured as a function of the cell culture cycle. The relative distribution of the enzymes between the 3000, 10,000 and 100,000 g pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per g fresh weight with time had a unique profile for each enzyme tested. Maximal yield of the proton translocating ATPase activity was obtained from cells in the middle logarithmic phase of growth, and from 50-90% of the activity was found in the 10,000 g pellet. The proton translocating ATPase activity was separable from NADPH cytochrome c reductase and cytochrome c oxidase on a sucrose gradient. Proton transport activity had a broad pH optimum (7.0-8.0), was stimulated by KCl with a Km of 5-10 mM, stimulation being due to the anion, Cl-, and not the cation, K+, and was not inhibited by vanadate; however, it was inhibited by NO3-. The activity is tentatively identified as the tonoplast ATPase.