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

被引:56
作者
SERRANO, A [1 ]
CORDOBA, F [1 ]
GONZALEZREYES, JA [1 ]
NAVAS, P [1 ]
VILLALBA, JM [1 ]
机构
[1] NATL UNIV CORDOBA, FAC CIENCIAS, DEPT BIOL CELULAR, E-14004 CORDOBA, SPAIN
关键词
D O I
10.1104/pp.106.1.87
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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-CB, and dye-ligand affinity chromatography in Blue-Sepharose CL-CB 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-ethylmaleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifruoroacetone, and the thiol reagent p-hydroxymercuribenzoate.
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页码:87 / 96
页数:10
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