Plasma membrane purification from roots of sunflower by phase partitioning.

Abstract

Plasma membrane vesicles were purified from the roots of sunflower (Helianthus annuus L. cv. Topflor) by aqueous polymer two-phase partitioning. The optimal conditions for separation were determined by systematic variation of the polymer concentration and salt composition. The phase system containing 6% (w/w) dextran T-500, 6% (w/w) polyethylene glycol 3350, 250 mM sucrose, 5 mM potassium phosphate, pH 7.8, without added salts proved to be the best. The ATPase activity had a pH optimum at 6.5 and it was stimulated by Mg2+, but not by Ca2+. The plasma membrane MgATPase activity was inhibited by vanadate but not by nitrate, an inhibitor of tonoplast ATPase. Only 10% of the microsomal protein was responsible for 36% of the total MgATPase activity. Moreover IDPase activity, a Golgi marker, appeared to be very low indicating the high purity of the preparation.