Proton-transport activity, sidedness, and morphometry of tonoplast and plasma-membrane vesicles purified by free-flow electrophoresis from roots of Lepidium sativum L. and hypocotyls of Cucurbita pepo L.

Large-scale preparations of highly purified tonoplast and plasma-membrane vesicles were obtained from roots (garden cress, Lepidium sativum L.) and shoots (etiolated zucchini hypocotyl, Cucurbita pepo L.) of representative dicotyledonous seedlings. When tonoplast-enriched fractions of cress roots were prepared by centrifugation and then subjected to free-flow electrophoresis a highly purified tonoplast fraction was obtained. This fraction from cress roots was characterized by morphometry of filipin-treated freeze-fractured preparations and by enzymology to be about 90% homogeneous. Using latency of nitrate-inhibited ATPase and H(+)-pumping as criteria we found that the majority of the tonoplast vesicles from both sources were oriented right(cytoplasmic)-side-out. Plasma-membrane vesicles were first purified by two-phase partitioning and then subjected to free-flow electrophoresis for further purification. From cress roots, the fraction of highest purity contained 89% plasma-membrane vesicles as judged by morphometry of filipin-treated, freeze-fractured preparations and by enzymology. From both sources, the major plasma-membrane subfraction in the upper phase after two-phase partitioning was shown to have the least electrophoretic mobility in free-flow electrophoresis and to be oriented right(extracytoplasmic)-side-out a slightly more mobile plasma-membrane subfraction was oriented inside-out and originated after freezing thawing from outside-out plasma-membrane vesicles.