Characterization and solubilization of residual redox activity in salt-washed and detergent-treated plasma membrane vesicles from spinach leaves

An NADH -hexacyanoferrate (III) oxidoreductase (N-HCF-OR) was purified from spinach leaf plasma membrane (PM) vesicles; detailed biochemical analyses, however, revealed that the purifed protein is an NADH-monodehydroascorbate oxidoreductase (N-MDA-OR) located on the cytoplasmic surface of the PM. Af ter removing all N-MDA-OP activity from the PM vesicles by consecutive trea tments with hypoosmotic shock, salt, and detergents, the remaining PM (the "stripped" PM, SPM) fraction contained about 50% of the protein and 15% of the N-HCF-OR activity of the original PM fraction. The highest redox activi ty (100%) of the SPM fraction was obtained with NADH as electron donor and hexacyanoferrate(III) (HCF) as electron acceptor, although redox activity c ould be measured also with ubiquinone-0 (23%), dichlorophenolindophenol (16 %), cytochrome c (9%), and Fe3+-EDTA (2%) as electron acceptors. The follow ing K-m values were obtained for the N-HCF-OR activity of SPM: K-m(NADH) = 66.5 +/- 3.8 mu M [with 200 mu M HCF(III)], Km[HCF(III)] = 11.1 +/- l.l mu M (with 150 mu M NADH). NAD(+) competitively inhibited the activity. Under special conditions, SB-16 (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) solubilized about 50% of the protein and mor e than 90% of the N-HCF-OR activity of the SPM fraction. Redox activity of the solubilized fraction with dichlorophenolindophenol as electron acceptor was 45% of that with HCF(III). The SB-16-solubilized fraction contained b- type cytochrome(s) which could be reduced by dithionite > ascorbate >> NADH . Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SB-16-solubilized SPM fraction revealed numerous polypeptides betwee n 17 and 95 kDa. Further purification steps are needed to match the redox a ctivities and spectrophotometric data to one or more of the polypeptides se en on the gel.