Comparison of the properties of plasmalemma vesicles purified from wheat roots by phase partitioning and by discontinuous sucrose gradient centrifugation

Abstract

Plasmalemma vesicles were purified from 7‐day‐old wheat (Triticum aestivum L. cv. Drabant) roots by a) discontinuous sucrose gradient (SG), b) aqueous polymer two‐phase partitioning (PP) and c) SG followed by PP (SG + PP). SG‐purified plasmalemma preparations were 2‐3 fold more contaminated with mitochondria (cytochrome c oxidase, EC 1.9.3.1) when compared to PP and (SG + PP)‐purified plasmalemma. The electrostatic surface properties, as measured by 9‐aminoacridine fluorescence, were similar in PP and (SG + PP)‐purified plasmalemma and different from SG‐purified. The latency of the MG‐ATPase measured with Triton X‐100 was 51, 81 and 82% for SG‐, PP‐ and (SG + PP)‐purified plasmalemma vesicles, respectively. The higher latency of the ATPase (and lower specific activity in the absence of Triton X‐100) in PP‐purified and (SG + PP)‐purified preparations was not due to an effect of PEG, since exposure of SG‐purified preparations to PEG either in the wash medium or in the ATPase assay medium did not change ATPase activity. It is concluded that SG‐purified plasmalemma vesicles are more contaminated than PP‐purified preparations and that the former are likely to be leaky. They are, therefore, less suitable for use in studies of transport across the plasmalemma.