Isolation Of H +-Transport-Competent Plasma Membrane Vesicles From Corn Roots By Discontinuous Sucrose Gradient Centrifugation: Effect of Membrane Protectant Agents

Abstract

Summary 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- l-phosphate, to the homogenizing and sucrose gradient media, 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 - > N0 3 - >> S0 4 - > IDA - and was therefore different from the sequences determined with plasma membrane vesicles isolated from other plant tissues. The lower effectiveness of N0 3 - 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.