ISOLATION OF H+-TRANSPORT-COMPETENT PLASMA-MEMBRANE VESICLES FROM CORN ROOTS BY DISCONTINUOUS SUCROSE GRADIENT CENTRIFUGATION - EFFECT OF MEMBRANE PROTECTANT AGENTS

Crude KI-washed plasma membrane vesicles isolated from corn roots were further purified on a discontinuous sucrose gradient. Due to the addition of the substrate analogue of phospholipase D, choline, and an inhibitor of the phosphatidic acid phosphatase, sodium glycerol-1-phosphate, to the homogenizing and sucrose gradient me dia, purified plasma membrane vesicles collected at the 30%/38% interphase conserved a high specific activity of H+-transport. In particular, the presence of choline seemed to be essential to stabilize the ATP-dependent H+-accumulation of membrane vesicles. The H+-transport activity was sensitive to vanadate only. The pH optimum of H+-transport was between 6.0 and 6.5. The H+-transport was highly stimulated by K+ without any lag phase, which was consistent with a direct action of K+ on the plasma membrane ATPase at the cytoplasmic vesicle surface facing the medium. The K+-stimulation of H+-transport was concentration dependent and increased up to 100 mM K+ without achieving saturation. The H+-transport activity depended on the presence of permeant anions. The sequence of anion stimulation followed the order of Br- > Cl- > NO3- >> SO4- > IDA(-) and was therefore different from the sequences determined with plasma membrane vesicles isolated from other plant tissues. The lower effectiveness of NO3- than Br- and Cl- in stimulating H+-translocation did not originate in tonoplast contamination but appeared rather to be related to genetic differences in corn varieties.