ACTIVE UPTAKE OF SUCROSE BY PLANT PLASMA-MEMBRANE VESICLES - DETERMINATION OF SOME IMPORTANT PHYSICAL AND ENERGETICAL PARAMETERS

The present paper investigates some characteristics of plant plasma membrane vesicles in relation to sucrose uptake. The plasma membrane vesicles purified by aqueous two-phase partitioning from sugar beet (Beta vulgaris L.) leaf microsomes had an average diameter of 291 +/- 13 nm and their internal volume was 2.2-mu-l (mg protein)-1. Although the vesicles were subjected to one freeze-thaw cycle for the purpose of storage, they retained mainly (78 %) their original right-side-out orientation as estimated either by labelling with peroxidase-labelled Concanavalin A or by measurements of ATPase latency. The use of higher concentrations of protecting agents [5 mM ethylenediaminetetraacetic acid (EDTA) and 2 mM dithiothreitol (DTT)] in the griding buffer led to an increase of sucrose uptake by the vesicles. Measurements of the two components (pH and electrical gradients) of the proton motive force (PMF) showed that a high PMF (-200 mV) was created under the experimental conditions used. The accumulation of sucrose which theoretically could be obtained by this PMF is much larger than the accumulation actually measured. Although the data support the conclusion that an active uptake of sucrose is driven by the PMF in plasma membrane vesicles from sugar beet leaves, they also stress some limits of this experimental system.