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Characterization of reconstituted plasma membrane H + ‐ATPase from maize roots
Author(s) -
Hsu AnFei C.,
Brauer David,
Tu ShuI
Publication year - 1989
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.1399-3054.1989.tb05476.x
Subject(s) - vanadate , atpase , chemistry , vesicle , chromatography , proton transport , membrane , enzyme , biochemistry , atp hydrolysis , hydrolysis , centrifugation
Corn ( Zea mays L.) plasma membranes from KI‐washed microsomal fractions were further purified by isopycnic sucrose density centrifugation. An examination of separated fractions indicated that vesicles with nitrate‐insensitive proton transport copurified with fractions containing vanadate‐sensitive ATPase activity. The ATPase in purified plasma membrane was reconstituted into liposomes by a detergent dilution technique using deoxycholate. The reconstituted ATPase exhibited characteristics similar to those of the native enzyme. However, reconstituted preparations showed an enhanced sensitivity to vanadate, a diminished phosphatase activity and a high specific rate of ATP‐dependent H + ‐transport. Apparent K i values of reconstituted and native enzymes with respect to vanadate were 20 and 50 μ M , respectively; the KJ value of the H+‐pumping of reconstituted ATPase was 30 μ M. The proton pumping of reconstituted vesicles could be discharged rapidly by p ‐trifluoromethoxyphenyl hydrazone (FCCP), hexokinase and vanadate. The hydrolysis of Mg‐ATP by both native and reconstituted ATPases obeyed simple Michaelis‐Menten plots with a K m between 0.5 and 0.6 m M. The reconstituted ATPase retained a pH profile similar to that of native enzyme with a maximum of pH 6.5.