Dissecting the Diphenylene Iodonium-Sensitive NAD(P)H:Quinone Oxidoreductase of Zucchini Plasma Membrane

Abstract

Quinone oxidoreductase activities dependent on pyridine nucleotides are associated with the plasma membrane (PM) i n ZUC-chini (Cucurbifa pepo L.) hypocotyls. I n the presence of NADPH, lipophilic ubiquinone homologs with up t o three isoprenoid units were reduced by intact PM vesicles with a K,,, of 2 t o 7 p M. Affinities for both NADPH and N A D H were similar (K, of 62 and 51 p ~ , respectively). Two NAD(P)H:quinone oxidoreductase forms were identified. The first, labeled as peak I i n gel-filtration experiments, behaves as an intrinsic membrane complex of about 300 kD, it slightly prefers N A D H over NADPH, it is markedly sensitive t o the inhibitor diphenylene iodonium, and it is active with lipophilic quinones. The second form (peak II) is an NADPH-preferring oxidoreductase of about 90 kD, weakly bound t o the PM. Peak II is diphenylene iodonium-insensitive and resembles, in many properties, the soluble NAD(P)H:quinone oxidoreductase that is also present i n the same tissue. Following purification of peak I, however, the latter gave rise t o a quinone oxidoreductase of the soluble type (peak II), based o n substrate and inhibitor specificities and chromatographic and electrophoretic evidence. It is proposed that a redox protein of the same class as the soluble NAD(P)H:quinone oxidoreductase (F. Sparla, C. Tedeschi, and P. Trost 11 9961 Plant Physiol. 1 1 2:249-258) is a component of the diphenylene iodonium-sensitive PM complex capable of reducing lipophilic quinones. ~ ~ Plant cells appear to contain severa1 types of NAD(P)H-dependent quinone oxidoreductases in addition to those of energy-conserving reactions of plastids and mitochondria. The purification of some of these plant proteins has been accomplished, but their biochemical characterization is still at an initial stage. Recently, NAD(P)H-QR from plant tissues was purified and characterized (Rescigno et al., 1995; Trost et al., 1995; Sparla et al., 1996). This plant oxidoreduc-tase represents a functional equivalent of animal DT-diaphorase, since it reduces short-chain quinones to quin-01s by two-electron donation without semiquinone intermediates, thereby resulting in enhanced quinone conjugation and low probability of formation of active oxygen species (Trost et al., 1995). However, a number of properties are different from animal-type DT-diaphorase, i.e. the NAD(P)H-QR contains FMN, it has a mass of about 90 kD with subunits of 21.4 kD (by MS), and the hydride transfer

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).…”

Naphthoquinone-Dependent Generation of Superoxide Radicals by Quinone Reductase Isolated from the Plasma Membrane of Soybean

“…Examples of this class are the NAD(P)H:quinone-acceptor oxidoreductases known as DT-diaphorases in animals (Lind et al, 1990;Ross, 1997). Functionally related flavoenzymes have also been found in plants (Spitsberg and Coscia, 1982;Luster and Buckhout, 1989;Valenti et al, 1990;Serrano et al, 1994;Rescigno et al, 1995;Trost et al, 1995Trost et al, , 1997Sparla et al, 1996Sparla et al, , 1998 (Iyanagi and Yamazaki, 1970;Lind et al, 1990;Sparla et al, 1996;Ross, 1997). A purified quinone oxidoreductase investigated by Trost et al (1997) was shown to be insensitive to DPI.…”