Localization of donor and acceptor sites of NADH dehydrogenase activities using inside‐out and right‐side‐out plasma membrane vesicles from plants

Askerlund, Per; Larsson, Christer; Widell, Susanne

doi:10.1016/0014-5793(88)80538-6

Cited by 52 publications

(26 citation statements)

References 25 publications

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“…The activity was recorded as A(A550-A600), and was determined ±0.015% (w/v) Triton X-100 with plasma membranes (1). The extinction coefficients used were 1 and 19 mM-' cm-' for ferricyanide and Cyt c, respectively.…”

Section: Western Immunoblotting Analysismentioning

confidence: 99%

“…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).…”

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confidence: 99%

“…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 of NADH-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 membranes (33,34).…”

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confidence: 99%

“…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.…”

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NADH-Ferricyanide Reductase of Leaf Plasma Membranes

Askerlund

Praly²,

Nakagawa³

et al. 1991

Plant Physiol.

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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-

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Section: Western Immunoblotting Analysismentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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NADH-Ferricyanide Reductase of Leaf Plasma Membranes

Askerlund

Praly²,

Nakagawa³

et al. 1991

Plant Physiol.

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“…NADH-acceptor oxidoreductase with either ferricyanide or Cyt c (Sigma; C 7752) as acceptor was assayed essentially as described earlier (3).…”

Section: Nadh-acceptor Oxidoreductasementioning

confidence: 99%

Sealed Inside-Out and Right-Side-Out Plasma Membrane Vesicles

Palmgren¹,

Askerlund²,

Fredrikson³

et al. 1990

Plant Physiol.

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Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of insideout vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H+ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% rightside-out vesicles, respectively, were obtained. ATPase activity and H+ pumping were both completely inhibited by vanadate (K, ; 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation.A typical feature ofbiological membranes is the asymmetric arrangement of constituents across the lipid bilayer. This asymmetry is absolute for proteins: integral proteins, which span the membrane, expose different regions on either side of This paper is dedicated to Professor Per-Ake Albertsson, the pioneer of aqueous polymer two-phase partitioning, on the occasion of his 60th birthday. the membrane, whereas peripheral proteins are bound to either surface of the membrane. For lipids, the asymmetry is rather relative than absolute, such that each lipid species usually only shows some enrichment to either half of the bilayer. This asymmetric, transverse organization of membrane constituents forms the basis for all the vectorial activities exerted by biological membranes, and is created through the asymmetric assembly of membranes (review, 22).The most useful approach for characterizing the asymmetric properties ofa membrane, including its vectorial activities, is to prepare sealed membrane vesicles of either orientation. With such preparations, each membrane surface can be probed selectively using impermeable agents, and transport in either direction can be measured as uptake into vesicles. The formation and subsequent separation of vesicles of opposite orientation was first achieved with the erythrocyte membrane through the pioneering work ofSteck et ...

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