Fig. 1.

Evaluation of vacuolar membrane (A, C) and plasma membrane (B, D) purity by enzymatic characterization and western blot analyses. During tonoplast preparation, total membranes were fractionated on a sucrose gradient and the resulting fractions were subjected to the analyses of marker enzyme activity (A) and blotted with antibodies specific for V-ATPase and PM H⁺-ATPase (C). Fraction 2 was used in the further assay since it was enriched in tonoplast vesicles of good yield and purity. In contrast, the fractionation of the total membranes on the two-phase system allowed two phases to be obtained, with the upper phase (fraction 1) enriched in pure plasma membranes (D). Marker enzyme activities confirmed the purity of the plasma membranes in fraction 1 (B).