Study of sidedness and tightness to H + of corn root plasmalemma vesicles: preparation of a fraction enriched in inside-out vesicles

Abstract

The latency of the Mg 2+-ATPase of corn root plasmalemma was measured using Triton X-100, Zwittergent 3–14, sodium dodecyl sulfate and lysophosphatidylcholine. Lysophosphatidylcholine was the only permeabilizing agent which did not inhibit the enzyme. It gave a higher latency than the other surfactants. The sidedness of plasmalemma vesicles obtained after sucrose gradient purification was estimated from: (i) the Mg 2+-ATPase latency; (ii) the fraction of Mg 2+-ATPase resistant to trypsin attack; (iii) the effect of the specific binding of concanavalin A on the microelectrophoretic mobility of the vesicles; and (iv) the separation of the vesicles by chromatography on a Sepharose-Con A column. These four kinds of independent data were consistent. They led to the conclusion that this membrane preparation contained only sealed vesicles (impermeable to MgATP), the majority of which (approx. 65%) were oriented right-side out. Addition of 0.25 M sucrose in the same way inhibited the MgATP hydrolysis measured with or without lysophosphatidylcholine, so that the latency of the activity remained unchanged. The inhibition was higher in the presence of 0.25 M inositol and trehalose, again without modifying the latency. Finally, this preparation contained no significant proportion of tightly sealed active vesicles, as indicated by the absence of quinacrine quenching after addition of MgATP. Tightly sealed vesicles, displaying quinacrine quenching, were obtained according to De Michelis and Spanswick ((1986) Plant Physiol. 81, 542–547). The sidedness in this vesicle preparation was the same as the previous one (approx. 65% right-side out). Washing with Triton X-100 described by Clement et al. ((1986) Physiol. Vég. 24, 25–35) was applied. The simultaneous measurements of quinacrine quenching and Mg 2+-ATPase latency suggested a general inversion of the vesicle sidedness (approx. 65% of inside-out vesicles) after 0.1% treatment with (w / v) Triton X-100. Furthermore, the total Mg 2+-ATPase activity was recovered in spite of protein loss of approx. 60%, and the specific activity increased by approx. 150% in the presence of lysophosphatidylcholine. In the absence of lysophosphatidylcholine, coupling of Mg 2+-ATPase activity with H + transport was indicated by a 30% increase of MgATP hydrolysis upon addition of gramicidin or NH 4Cl.