Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants

Abstract

A simplified method for the isolation of a plasma membrane-enriched fraction from plants utilizing an aqueous two-polymer phase system is outlined. Mainly, the plant used was Orchard grass (Dactylis glomerata L.). The two-phase system consisted of 5.6% (w/w) of dextran T500 and 5.6% (w/w) of polyethyleneglycol 4000 in 0.5 molar sorbitol-15 millimolar Tris-maleate (pH 7.3), and 30 millimolar NaCl. In this system, the plasma membranes and the other membranes were preferentially partitioned into the top phase and into the lower phase, respectively. The purity of the isolated plasma membrane was sufficiently high even after a single partition (i.e. about 85% purity) and more than 90% purity was obtained after repeating the partition in a newly prepared lower phase. The plasma membrane was identified with the aid of phosphotungstic acid-chromic acid stain and the association of vanadate-sensitive Mg(2+)-ATPase. The plasma membrane-associated ATPase had a pH optimum at 6.5 and showed a high specificity for Mg(2+) and ATP. KCl stimulation was low (6% stimulation) at the pH optimum, but a relatively high stimulation (23%) occurred at pH 5.5. This method for plasma membrane isolation may be applicable to a wide variety of plants and plant tissue including green leaves.