Characterization of reconstituted plasma membrane H+-ATPase from maize roots |
| |
Authors: | An-Fei C Hsu David Brauer Shu-I Tu |
| |
Institution: | U.S. Dept of Agriculture, ARS, Eastern Regional Research Center, Plant and Soil Biophysics Research Unit, 600 East Mermaid Lane, Philadelphia, PA 19118, USA. |
| |
Abstract: | 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 Ki 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 Km 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. |
| |
Keywords: | Corn roots H+-ATPase maize membrane reconstitution plasma membrane proton transport |
|
|