Preparation of Membrane Vesicles Enriched in ATP-Dependent Proton Transport from Suspension Cultures of Tomato Cells

Abstract

Membranes enriched in ATP-dependent proton transport were prepared from suspension cultures of tomato cells (Lycopersicon ecukatum Mill cv VF36). 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 3,000, 10,000, and 100,OOOg pellets remained the same throughout the cell culture cycle, but yield of total activity and activity per gram 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 to 90% of the activity was found in the 10,OOOg 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 (7.04.0), was stimulated by KCI with a K. of 5 to 10 millimolar, stimulation being due to the anion, Cl-, and not the cation, K, and was not inhibited by vanadate, but was inhibited by N03-. The activity is tentatively identiffied as the tonoplast ATPase.Since methods are available to obtain sealed vesicles from tonoplast or plasma membrane of plant tissues and to measure ATP-dependent proton transport in vitro (1-5, 10, 13, 16), it is reasonable to contemplate using the isolated vesicles to investigate the response of the plasma membrane or tonoplast to environmental stress. We investigated the effect of chilling temperature on suspension cultures of tomato cells (6) to characterizing the effect of assay conditions on enzyme activity. MATERIALS AND METHODSCell Culture. Methods were as previously described (6). Callus was initiated from roots (growth curve experiment) or hypocotyls (all other experiments) of aseptic tomato seedlings (Lycopersicon esculentum Mill cv VF36) germinated on agar. Suspension cultures were initiated 2 months to 1 year after initiation of callus, and maintained for up to 1 year prior to the experiments shown. The cultures were maintained in 200 ml of medium in 500 ml flasks on a reciprocating shaker at 28°C. The medium (6) Preparations enriched in sealed vesicles were prepared by centrifugation onto a cushion of 10% 70 kD Dextran (4, 18).

Acclimation to Low Temperature by Microsomal Membranes from Tomato Cell Cultures

“…Suspension cultures provide a source of large quantities of membranes while making it possible to avoid or accurately monitor the effects of age and differentiation on membrane composition. Vesicles, capable of ATP-dependent proton transport, have been isolated from tomato cells and characterized (9). The characteristics of the proton-translocating ATPase resemble those of the tonoplast ATPase of red beet (1,23).…”

“…Proton transport was assayed as ATP-dependent quench of fluorescence of acridine orange (9 (27). Aliquots (0.5 ml) of the reaction mixture were removed at timed intervals, immediately placed in a mixture of 1.5 ml of 12% TCA plus 0.5 ml of 3.75% ammonium molybdate in 3 N H2SO4.…”