PURIFICATION AND CHARACTERIZATION OF 2 DISTINCT NAD(P)H DEHYDROGENASES FROM ONION (ALLIUM-CEPA L) ROOT PLASMA-MEMBRANE

Highly purified plasma membrane fractions were obtained from onion (Al lium cepa L.) roots and used as a source for purification of redox pro teins. Plasma membranes solubilized with Triton X-100 contained two di stinct 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-CB, and dye-ligand affinity chromatography in Blue-Sepharose CL-CB after biospecific elut ion with NADH. Dehydrogenase I consisted of a single polypeptide of ab out 27 kD and an isoelectric point of about 6. Dehydrogenase II was pu rified from the DEAE-unbound fraction by chromatography in Blue-Sephar ose CL-6B and affinity elution with NADH. Dehydrogenase II consisted o f a single polypeptide of about 31 kD and an isoelectric point of abou t 8. Purified dehydrogenase I oxidized both NADPH and NADH, although h igher rates of electron transfer were obtained with NADPH. Maximal act ivity was achieved with NADPH as donor and juglone or coenzyme Q as ac ceptor. Dehydrogenase II was specific for NADH and exhibited maximal a ctivity with ferricyanide. Optimal pH for both dehydrogenases was abou t 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltri fluoroacetone, and the thiol reagent N-ethylmaleimide. A strong inhibi tion of dehydrogenase II was obtained with dicumarol, thenoyltrifruoro acetone, and the thiol reagent p-hydroxymercuribenzoate.