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Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Memon, Abdul Razaque
Sommarin, Marianne
Kylin, Anders
Plasmalemma was isolated from the roots of 2‐week‐old cucumber plants (Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two‐phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K+, Mg2+‐ATPase) was 14‐ to 17‐times higher in the upper (PEG‐rich) than in the lower (Dextran‐rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC, and antimycin A‐resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X‐100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right‐side‐out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg2+. This optimum was shifted to pH 5.8 after addition of K+. K+ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N‐dicyclohexylcarbodiimide and sodium vanadate, with K+ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO−3, azide and oligomycin. No Ca2+‐ATPase was detected, and even as little as 0.05 mM Ca2+ inhibited the Mg2+‐ATPase activity.