Purification and characterization of the NADPH-cytochrome <i>P</i>-450 (cytochrome <i>c</i>) reductase from higher-plant microsomal fraction

Benveniste, Irène; Gabriac, B; Durst, Francis

doi:10.1042/bj2350365

Cited by 75 publications

(39 citation statements)

References 47 publications

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“…Purification of NADPH-Cyt P-450 Reductase NADPH-Cyt P-450 reductase (fpl) from Jerusalem artichoke (Helianthus tuberosus) was purified from solubilized microsomes as described (2).…”

Section: Methodsmentioning

confidence: 99%

“…The solution of purified immunoglobulins could be diluted up to 10-fold. Cyt P-450 monooxygenase activities and Cyt c reduction were then measured as described (1,2 During the course of these immunochemical studies, we achieved the purification of three closely related isoforms of the reductase (4,17). These isoforms are partly resolved and visualized on 7.5% SDS-PAGE.…”

Section: Selection Of Clones Inhibiting the Activity Of Nadph-cyt P-4mentioning

confidence: 99%

“…From Jerusalem artichoke (Helianthus tuberosus) tubers we purified a plant NADPH-Cyt P-450 reductase (2). This enzyme preparation, which migrated as a single broad band (82 kD) on 12.5% SDS-PAGE, was used to raise polyclonal antibodies (3).…”

mentioning

confidence: 99%

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Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Lesot

Benveniste²,

Hasenfratz³

et al. 1992

Plant Physiol.

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Monoclonal antibodies (mAbs) against a plant NADPH-cytochrome P-450 (Cyt P-450) reductase from Jerusalem artichoke (Helianthus tuberosus) tuber were prepared. These antibodies were produced by hybridoma resulting from the fusion of spleen cells from a rat immunized with a purified preparation of the reductase and mouse myeloma cells. The mAbs thus obtained were screened for their interaction with the reductases, first in western dots and then in blots, and for their ability to inhibit the NADPHcytochrome c (Cyt c) reductase activity from Jerusalem artichoke microsomes. Among the 11 clones giving a positive response on western blots, only 6 were also able to inhibit microsomal NADPHCyt c reductase activity, and the microsomal Cyt P-450 monooxygenase activities dependent upon electrons transferred by the reductase. Thus, two families of mAbs were characterized: a family of mAbs that interact with epitopes of the reductase implicated in the reduction of Cyt P-450 by NADPH (binding sites for NADPH, flavin mononucleotide, flavin adenine dinucleotide, and Cyt P-450), and a structural family, whose members recognize epitopes outside the active site of the reductases. These mAbs specifically recognize the reductase, and all of them interact with all of the isoforms, indicating that important primary or secondary structural analogies exist between the isoforms, not only at the active site, but also at the level of epitopes not directly associated with catalytic activity.markable by the presence of both FAD2 and FMN prosthetic groups, exists as a single form in animals (23). From Jerusalem artichoke (Helianthus tuberosus) tubers we purified a plant NADPH-Cyt P-450 reductase (2). This enzyme preparation, which migrated as a single broad band (82 kD) on 12.5% SDS-PAGE, was used to raise polyclonal antibodies (3). Western blotting of the reductase after 7.5% SDS-PAGE showed that the reductase preparation was composed of three proteins of very close molecular mass: 80, 82, and 84 kD. To get insight into the function and immunological relationships among these proteins, we set out to produce mAbs against the reductase preparation. A similar approach had produced mAbs specific for hepatic Cyt P-450 isoforms from rats treated with different inducers (21).Here we present the characterization of two families of mAbs we have obtained, and we discuss the relationship between the isoforms of Jerusalem artichoke NADPH-Cyt P-450 reductase. MATERIALS AND METHODSPurification of NADPH-Cyt P-450 Reductase NADPH-Cyt P-450 reductase (fpl) from Jerusalem artichoke (Helianthus tuberosus) was purified from solubilized microsomes as described (2).The microsomal Cyt P-450-dependent monooxygenases are multienzyme complexes consisting essentially of the hemoprotein Cyt P-450 and the flavoprotein NADPH-Cyt P-450 reductase (24). In plants, different isoforms of Cyt P-450 are implicated in essential biosynthetic pathways such as the production of lignin, at the level of cinnamate hydroxylation (1) and ferulate hydroxylation (11); the biosynthe...

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“…Purification of NADPH-Cyt P-450 Reductase NADPH-Cyt P-450 reductase (fpl) from Jerusalem artichoke (Helianthus tuberosus) was purified from solubilized microsomes as described (2).…”

Section: Methodsmentioning

confidence: 99%

Section: Selection Of Clones Inhibiting the Activity Of Nadph-cyt P-4mentioning

confidence: 99%

See 1 more Smart Citation

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Lesot

Benveniste²,

Hasenfratz³

et al. 1992

Plant Physiol.

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“…The reductase was subsequently purified to homogeneity on a column of 2',5'-ADP-Sepharose 48 (Pharmacia), as previously reported (Sibbesen et al, 1995), and concentrated to approximately 15 units/mL (Benveniste et al, 1986).…”

Section: Lsolation Of the Nadph-p450 Oxidoreductasementioning

confidence: 99%

“…L11862). The barley j3 subunit has a predicted molecular mass of 54 kD (Berkelman et al, 1994). A monoclonal antibody raised against the /3 subunit of the vacuolar ATPase from oat roots was provided by Dr. Heven Sze.…”

Section: Purification Of P450oxmentioning

confidence: 99%

Isolation and Reconstitution of Cytochrome P450ox and in Vitro Reconstitution of the Entire Biosynthetic Pathway of the Cyanogenic Glucoside Dhurrin from Sorghum

Kahn¹,

Bak²,

Svendsen³

et al. 1997

Plant Physiology

123

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A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside β-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-α--dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled.

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Biotransformation ofs-Triazine Herbicides and Related Degradation Products in Liquid Cultures by the White Rot FungusPhanerochaete chrysosporium

Mougin

Laugero²,

Asther³

et al. 1997

Pestic. Sci.

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The ability of the white rot basidiomycete Phanerochaete chrysosporium to transform s‐triazine herbicides has been investigated in laboratory experiments. The chlorinated metabolites formed during atrazine N‐dealkylations were not further transformed by the fungus, whereas hydroxy‐atrazine was converted to an unknown product. P. chrysosporium was also able to carry out the N‐dealkylation of the herbicides simazine, propazine and terbuthylazine. Herbicide metabolism was not supported by purified peroxidases. The highest rates of herbicide N‐dealkylation were obtained in liquid cultures maintained under moderate temperature allowing a long mycelium growing phase. Atrazine transformation was found to be supported by the mycelium, which contained significant amounts of microsomal cytochrome P450. Herbicide N‐dealkylation was decreased in the presence of 1‐aminobenzotriazole, in agreement with the involvement of P450 monooxygenases in atrazine metabolism. © 1997 SCI.

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Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction

Cited by 75 publications

References 47 publications

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Production and Characterization of Monoclonal Antibodies against NADPH-Cytochrome P-450 Reductases from Helianthus tuberosus

Isolation and Reconstitution of Cytochrome P450ox and in Vitro Reconstitution of the Entire Biosynthetic Pathway of the Cyanogenic Glucoside Dhurrin from Sorghum

Biotransformation ofs-Triazine Herbicides and Related Degradation Products in Liquid Cultures by the White Rot FungusPhanerochaete chrysosporium

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