Topography of the 27- and 31-kDa electron transport proteins in the onion root plasma membrane.
Plasma membranes purified from onion roots contain two distinct NAD(P)H-dehydrogenases of 27 and 31 kDa that differ in their physicochemical properties, substrate specificities and inhibitors sensitivities. The 27-kDa enzyme used both NADH and NADPH as electron donors. The 31-kDa enzyme was fully specific for NADH and accounted for the bulk of NADH-ferricyanide oxidoreductase. We have used NADPH- and NADH-ferricyanide oxidoreductase activities as markers for investigating the orientation of the 27- and 31-kDa enzymes at the plasma membrane, respectively. These activities were assayed in right-side-out vesicles isolated by two-phase partition, inside-out vesicles obtained by treatment with the detergent Brij 58 and membranes permeabilized with Triton X-100. Upon addition of Brij 58 to right-side-out plasma membrane vesicles, both NADPH- and NADH-ferricyanide oxidoreductases were activated to the same degree as the plasma membrane H(+)-ATPase. Redox activities were similar when measured in the presence of either Brij 58 or Triton X-100. Our results demonstrate that both enzymes expose their catalytic sites toward the cytoplasmic side of the plasma membrane.
D000490:Allium; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D002463:Cell Membrane Permeability; D002592:Cetomacrogol / Q000494:pharmacology; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D004579:Electron Transport; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010084:Oxidation-Reduction; D018517:Plant Roots / Q000201:enzymology*
D003902:Detergents; D009243:NAD; D009249:NADP; D017830:Octoxynol; D002592:Cetomacrogol; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase