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

Date

1987-2

Author

Memon, Abdul Razaque
Sommarin, Marianne
Kylin, Anders

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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 1.9.3.1, 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.

Subject Keywords

Plant Science, Genetics, Cell Biology, Physiology, General Medicine

URI

https://hdl.handle.net/11511/52175

Collections

Department of Biology, Article