NADH-Ferricyanide Reductase of Leaf Plasma Membranes

Abstract

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-dia-

Reconstitution and Characterization of a Calmodulin-Stimulated Ca2+-Pumping ATPase Purified from Brassica oleracea L.

“…8). This is different from the purified and reconstituted Ca2+-ATPase from animal plasma membranes, which shows a significantly higher affinity for Ca2" in the presence of CaM [Km (Ca2") 1 (Fig. 8), resembling the case in animals (9).…”

Soluble and membrane‐associated nitrate reductases in the dinoflagellate Peridinium gatunense

“…Yet, some association of NR with membranes occurred with all buffers, as shown by Triton X-114 phase separation, during which only the more hydrophobic mNR remained in the detergent phase. For plant plasma membrane preparations, weak binding of NR to membranes has already been reported by Askerlund et al (1991) who even doubted the existence of a real pmNR. Nevertheless, Sto$ hr et al (1993), De Marco et al (1994) and Meyerhoff et al (1994) proved the existence of pmNR by different approaches.…”

Transmembrane electron transport in ascorbate‐loaded plasma membrane vesicles from higher plants involves a b‐type cytochrome

“…The tissue homogenisation buffer was supplemented with 100 mM sodium ascorbate to include high concentrations of the reductant inside the plasma membrane vesicles [8,9]. The membrane-impermeable electron acceptors, ferricyanide and cytoehrome c,were rapidly reduced by the aseorbate-loaded membranes ( Fig.…”

“…Recently a method became available for the prepara- tion of ascorbate-loaded plasma membrane vesicles, capable of electrogenic transmembrane electron transport [8,9]. These membrane fractions provide a tool to investigate the possible involvement of the high potential b-type cytochrome in trans-plasma membrane electron transport in plants.…”