NADH-Ferricyanide Reductase of Leaf Plasma Membranes1

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b5 reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas IgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and rightside-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propanesulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADHferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b5 reductase). Isolated plasma membranes of high purity show relatively high NAD(P)H-(acceptor) oxidoreductase [NAD(P)H-diaSupported in part by grants from the Swedish Natural Science Research Council and the Carl Tesdorpf Foundation. 2 Present address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford, OX 1, 3RB, UK. phorase] activities with different electron acceptors (1, 3, 4, 7, 8, 24). It has been suggested that these activities are due to redox systems capable of transferring electrons from cytoplasmic donors to electron acceptors in the apoplast. One such transplasma membrane electron transport system is thought to be induced in roots of nongraminaceous plants during iron deficiency and to be involved in the reduction of Fe3" to Fe2+ for uptake. Another system is thought to be constitutively present in all plant cells (for reviews, see 27, 28). Recently, Buckhout et al. (7) showed that NADH(but not NADPH-) (acceptor) oxidoreductase activities are induced in tomato root plasma membranes during Fe-deficiency, indicating that NADH is the donor for the inducible transplasma membrane electron transport system. An NAD(P)H-(acceptor) oxidoreductase has been purified from corn root plasma membranes (24, 25). Leaf cells also seem to possess a transplasma membrane redox system, since carefully washed leaf segments from both oat and sugar beet can reduce added ferricyanide3 (2, 12). By using inside-out and right-side-out plasma membrane vesicles from sugar beet leaves, however, we have shown that both donor and acceptor sites of the NADH-ferricyanide reductase are located on the cytoplasmic surface of the plasma membrane and that a possible transplasma membrane electron transport would constitute only a very minor proportion of the activity (1). Furthermore, right-side-out vesicles of sugar beet leafplasma membrane loaded with an NADH-generating system do not support reduction of external ferricyanide (2). The location of both donor and acceptor sites on the cytoplasmic surface (1), together with spectrophotometric data (3), led us (1) to suggest that NADH-Cyt b5 reductase (EC 1.6.2.2) was responsible for the major part of the NADHferricyanide reductase activity in leaf plasma membranes. Another possibility is that this activity is due to a plasma membrane-bound form ofNADH-NR (EC 1.6.6.1), since NR can reduce ferricyanide (9, 10, 29) and was recently suggested to be an integral component of barley and corn root plasma 3 Abbreviations: ferricyanide, K3[Fe(CN)6]; NR, nitrate reductase (EC 1.6.6.1); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio] 1propanesulfonate; FPLC, fast protein liquid chromatography; TLCK, N,a-p-tosyl-L-lysine chloromethyl ketone.