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 1.) 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-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, thenoyltrifluoroacetone, and the thiol reagent p-h ydroxymercuribenzoate.

Identification of New Intrinsic Proteins in Arabidopsis Plasma Membrane Proteome

“…The Cyt b5 isoform found in this study, among at least six different isoforms encoded by the Arabidopsis genome, was localized in the PM (this work), whereas the other isoforms are found in different cell compartments (57,58). In agreement with our data, FMN-reductases, known to participate in cell detoxication during oxidative stress, have been shown to be strongly bound to the PM (95,96).…”

Auxin-Responsive Genes AIR12 Code for a New Family of Plasma Membrane b-Type Cytochromes Specific to Flowering Plants

“…quinone reductases; Serrano et al, 1994;Trost et al, 1997;Schopfer et al, 2008;and MDA reductase;Bérczi and Møller, 1998), b-type cytochromes Bérczi et al, 2003;Preger et al, 2005), and peroxidases (Mika et al, 2008). Moreover, molecular biology approaches led to the discovery of flavocytochromes of the FRO family that use cytosolic pyridine nucleotides to reduce apoplastic ferrichelates (Robinson et al, 1999).…”

NAD(P)H:(Quinone-Acceptor) Oxidoreductase of Tobacco Leaves Is a Flavin Mononucleotide-Containing Flavoenzyme

“…According to some reports (Luster and Buckhout, 1989;Serrano et al, 1994), the plant root plasma membrane contains dehydrogenases with 27-kD subunits with properties of the same general protein type as NAD(P)H-QR, e.g. they show high NAD(P)H-dependent activity with hydrophilic quinones but low activity with Cyt c. The NAD(P)H-QR of corn root plasma membrane was stimulated by added FMN, so this flavin was thought to be a weakly bound enzyme cofactor (Luster and Buckhout, 1989).…”

“…However, their subunits appear to vary in size depending on the species (Chauveau and Lance, 1991;Luethy et al, 1991Luethy et al, ,1995Rasmusson et al, 1993). Plant plasma membrane also contains an NAD(P)Hdependent quinone oxidoreductase of 27-kD subunits (Luster and Buckhout, 1989;Serrano et al, 1994), the function of which has not yet been elucidated.…”

“…dicumarol is relatively ineffective as an inhibitor (Guerrini et al, 1987). NAD(P)H-QR occurs mainly in the soluble cell fraction, although NAD(P)Hdependent duroquinone reductase activities are also found in the mitochondria, microsomes, and plasma membrane (Asard et al, 1987;Luster and Buckhout, 1989;Valenti et al, 1990;Serrano et al, 1994;Rescigno et al, 1995).…”