Purification of a NADH-ferricyanide reductase from plant microsomal membranes with a zwitterionic detergent

Galle, Anne-Marie; Bonnerot, Claire; Jolliot, Alain; Kader, Jean‐Claude

doi:10.1016/0006-291x(84)91219-1

Cited by 29 publications

(15 citation statements)

References 16 publications

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“…A microsomal fraction from potato (Solanum tuberosum L. cv Bintje) tubers was prepared as described previously in Galle et al (13).…”

Section: Preparation Of Membranesmentioning

confidence: 99%

“…Antibodies against NADH-ferricyanide reductase purified from potato tuber microsomes as described previously (13) were raised in female New Zealand white rabbits. The rabbits were injected subcutaneously with 100 Ag of purified protein mixed 1:1 (v/v) with Freund's complete adjuvant.…”

Section: Preparation Of Antibodiesmentioning

confidence: 99%

“…The specificity of the antibodies raised against NADHferricyanide reductase purified from potato tuber microsomes (13) was investigated by immunoblotting with pure NADHferricyanide reductase and with a microsomal fraction from potato tubers (Fig. 1).…”

Section: Specificity Of Antibodies Raised Against Potato Tuber Microsmentioning

confidence: 99%

“…In the present paper we use antibodies against potato tuber microsomal NADH-ferricyanide reductase (tentatively identified as NADH-Cyt b5 reductase; see "Discussion" and ref. 13) and NR, respectively, to determine the nature of the NADH-ferricyanide reductase activity of leaf plasma membranes.…”

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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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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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“…A microsomal fraction from potato (Solanum tuberosum L. cv Bintje) tubers was prepared as described previously in Galle et al (13).…”

Section: Preparation Of Membranesmentioning

confidence: 99%

Section: Preparation Of Antibodiesmentioning

confidence: 99%

Section: Specificity Of Antibodies Raised Against Potato Tuber Microsmentioning

confidence: 99%

mentioning

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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“…Galle et al (5) have recently purified an NADH-ferricyanide reductase from potato tuber that is apparently involved in the desaturation of oleate to form linoleic acid in higher plants. Two mitochondrial forms of a reductase have also been isolated by Klein and Burke (8), the first of which contained two peptide fragments and was specific for NADH whereas the second was apparently comprised of five polypeptides, displayed a broad pH maxima, and utilized both NADH and NADPH.…”

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confidence: 99%

Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Jollie

Sligar²,

Schuler³

1987

Plant Physiol.

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In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b5 and NADH-cytochrome b5 reductase from the microsomal fraction of Pisum sativum. The cytochrome b5 component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The aminoterminal amino acid sequence of the cytochrome b5 component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.The microsomal electron transport systems of mammalian tissue play important roles in a variety of oxidative reactions including fatty acid desaturation and the support of mixed function oxidative biotransformations catalyzed by the Cyt P-450 monooxygenases. Reductive reactions of the latter class involve NADPH dehydrogenation by a flavoprotein, NADPH-Cyt P-450 reductase, containing both FAD and FMN prosthetic groups, followed by sequential one electron tansfers to the cytochrome P450 heme center. In addition, some P450 biotransformations can be mediated by electron transfer from a short redox transfer chain which links to the dephosphorylated form of pyridine nucleotide, NADH. This system includes NADH-Cyt b5 reductase, a flavoprotein with a single FAD prosthetic group, and the heme protein Cyt b5. Both the NADPH and NADH flavoprotein reductases as well as Cyt b5 have been purified to homogeneity from a variety of mammalian microsomal fractions and have received intensive study during the past decade (20,23,24). Both of these proteins of the NADH linked chain are amphipathic (15) in nature, consisting oftwo definite domains, a water soluble catalytic portion and a hydrophobic fragment that binds the protein to the microsomal membrane. The catalytic domain can be separated from the holenzyme by limited proteolytic digestion ofthe microsomal fraction (9, 21). Alternately, the intact enzyme can be obtained by detergent solubilization (16,20,24 Unfortunately, much less is known about the corresponding electron transport and P-450 monoxygenase systems from plant tissue. Galle et al. (5) have recently purified an NADH-ferricyanide reductase from potato tuber that is apparently involved in the desaturation of oleate to form linoleic acid in higher plants. Two mitochondrial forms of a reductase have also been isolated by Klein and Burke (8), the first of which contained two peptide fragments and was specific for NADH whereas the second was apparently comprised of five polypeptides, displayed a broad pH maxima, and utilized both NADH and NADPH. Efforts at the isolation of a Cyt P-450 reductase from the microsomal fraction of higher plants began with the partial pur...

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Genetic Manipulation of the Fatty Acid Composition of Plant Lipids

Somerville

Browse

1988

Opportunities for Phytochemistry in Plant Biotechnology

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Purification of a NADH-ferricyanide reductase from plant microsomal membranes with a zwitterionic detergent

Cited by 29 publications

References 16 publications

NADH-Ferricyanide Reductase of Leaf Plasma Membranes

NADH-Ferricyanide Reductase of Leaf Plasma Membranes

Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Genetic Manipulation of the Fatty Acid Composition of Plant Lipids

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Purification of a NADH-ferricyanide reductase from plant microsomal membranes with a zwitterionic detergent

Cited by 29 publications

References 16 publications

NADH-Ferricyanide Reductase of Leaf Plasma Membranes

NADH-Ferricyanide Reductase of Leaf Plasma Membranes

Purification and Characterization of Microsomal Cytochrome b5 and NADH Cytochrome b5 Reductase from Pisum sativum

Genetic Manipulation of the Fatty Acid Composition of Plant Lipids

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Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum