title: Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.
abstract: Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate.
authors.affiliation: Departamento de Biologia Celular, Facultad de Ciencias, Universidad de Cordoba, Avda. San Alberto Magno s/n, E-14004-Cordoba, Spain.
authors.forename: A.
authors.initials: A
authors.lastname: Serrano
authors.forename: F.
authors.initials: F
authors.lastname: Cordoba
authors.forename: J. A.
authors.initials: JA
authors.lastname: Gonzalez-Reyes
authors.forename: P.
authors.initials: P
authors.lastname: Navas
authors.forename: J. M.
authors.initials: JM
authors.lastname: Villalba