Plasmalemma isolation from cultured cells and roots of grapevine by aqueous two phase partition

Abstract

Plasma membrane fractions were isolated from grapevine cell culture (cv. Narancsízú crown gall tissue induced by Agrobacterium tumefaciens K 308) and grapevine roots (cv. Ezerjó and Leányka) by partitioning of microsomal fractions in a 6% dextran-polyethyleneglycol two-phase system. The presence of 250 mM KI in the homogenization buffer resulted in membrane vesicles free of loosely-bound proteins. The purification processes were monitored by measurement of Mg 2+-ATPase, cytochrome c oxidase and latent IDPase activities during a six-step counter current distribution. The pH dependence of Mg 2+-ATPase activity, K + stimulation, VO 4 3− inhibition and effects of different anions (NO 3 −, Cl −, SO 4 2−) were tested. These results indicated that plasma membrane (PM) vesicles have been separated into the upper phase, whereas mitochondrial, Golgi and tonoplast membranes appeared in the lower phase. The latent ATPase activity of the plasma membrane was about five times higher in PM preparation from cell culture than from roots. The properties of ATPase activities of cell culture and roots showed many similarities: pH dependence, K + stimulation at acidic pH, 50–60% vanadate inhibition, divalent cation activation: Mg 2+ > Mn 2+ > Co 2+ > Cu 2+ ⪢ Zn 2+ > Ca 2+. ATP was the best substrate in both cases; the order of substrate affinity was as follows: ATP ⪢ UTP, IDP ⪢ ADP > CTP, GTP, pNPP ⪢ AMP for cell culture and ATP > IDP ⪢ CTP, GTP ⪢ UTP, pNPP > AMP for the roots. The purification process was more effective with cell culture as compared to the roots.