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

Abstract

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...

Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1

“…The genes encoding oxidoreductases from plants have been cloned from several plant species and have been sequenced. They show a high degree of sequence similarity with the animal oxidoreductase (30). Apparently, the mammalian and plant oxidoreductases are similar enough that a human P450 can interact with a tobacco oxidoreductase.…”

Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

“…C4H activity was measured (26) by using [3][4][5][6][7][8][9][10][11][12][13][14] C]cinnamate as substrate [53.9 mCi͞mmol (1 Ci ϭ 37 GBq), Isotopchim, Ganagobie, France).…”

“…All known plant cytochrome P450 monooxygenase reactions depend on the associated activity of an NADPH:cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) that catalyzes the transfer of electrons from NADPH via FAD and FMN to the prosthetic heme group of the P450 protein. Although for animal systems detailed characteristics including structural properties of CPRs have been reported (4), only few of these enzymes have been purified and cloned from plant sources (5)(6)(7)(8).…”

“…In accord with their membrane-associated functions, CPRs are localized in the endoplasmic reticulum and frequently have been used as a marker for this subcellular fraction (11). Although mammals and yeast contain only a single CPR (9,12), two or even three isoforms each have been described for two plant species, Helianthus tuberosus (7) and Arabidopsis thaliana (8). However, the physiological relevance of the occurrence of multiple CPRs in these plants is unknown.…”

The NADPH-cytochrome P450 Reductase Gene from Gibberella fujikuroi Is Essential for Gibberellin Biosynthesis

“…In contrast to the many different cytochrome P450 monooxygenases that can be found in a single species, only one CPR-encoding gene is found in most organisms. Exceptionally, certain plants (22)(23)(24) and some zygomycetes (25) possess two or three CPRs, although the physiologi-* This work was supported by the Deutsche Forschungsgemeinschaft (Tu101/9-1) and Fondo Nacional de Desarrollo Cientifico y Tecnologico Grant 1020140. Rothamstead Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.…”