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Partial purification of plant plasma membrane K + , Mg 2+ ‐ATPase by ion exchange chromatography
Author(s) -
Bérczi Alajos,
Morré D. James
Publication year - 1992
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.1399-3054.1992.tb04724.x
Subject(s) - membrane , chemistry , chromatography , vesicle , atpase , elution , ion exchange , ionic strength , enzyme , aqueous solution , biochemistry , ion , organic chemistry
A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H + ‐pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two‐phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non‐ionic detergent (either Triton X‐100 or C 12 E 8 ). The Mg‐ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl‐eluted fractions contained a Mg‐ATPase which showed the characteristics of Mg‐ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg −1 min −1 when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.