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Plasmalemma from the roots of cucumber: Isolation by two‐phase partitioning and characterization
Author(s) -
Memon Abdul Razaque,
Sommarin Marianne,
Kylin Anders
Publication year - 1987
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1987.tb04281.x
Subject(s) - vanadate , oligomycin , sodium azide , chemistry , atpase , biochemistry , polyethylene glycol , peg ratio , enzyme , chromatography , finance , economics
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 + , Mg 2+ ‐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 Mg 2+ . 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 Ca 2+ ‐ATPase was detected, and even as little as 0.05 m M Ca 2+ inhibited the Mg 2+ ‐ATPase activity.