Preparation of corn root plasmalemma with low Mg-ATPase latency and high electrogenic H+ pumping activity after phase partitioning

Abstract

Crude plasma membranes of corn (Zea mays l.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H+-transport, was purified by phase partitioning. The percentage of inside-out vesicles (10%) was determined from the Mg-ATPase latency, revealed with lysophosphatidylcholine. A Triton X-100 treatment described previously (JP Grouzis, R Gibrat, J Rigaud, C Grignon 1987 Biochim Biophys Acta 903: 449-464) was applied to phase-partitioned plasma membranes. The percentage of catalytic sites freely accessible to Mg-ATP increased to 50% after Triton X-100 treatment. Treated vesicles remained capable of electrogenic H+-pumping, as demonstrated by MgrATP-dependent quinacrine fluorescence quenching and oxonol absorbance shift. As expected from the large increase of the catalytic sites accessibility, increases of the dye responses were observed. Concanavalin A binding was estimated from microelectrophoretic measurements of individual vesicles. Statistical analysis of concanavalin A binding and Mg-ATPase latency suggest that treated membranes have lost their asymmetric structure.