Rat NADPH-P450 Reductase Research Articles

Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP+ in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni2+-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis.

To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs. CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H2O2 and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates. CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

Functional characterization of steroid hydroxylase CYP106A1 derived from Bacillus megaterium.

In this study, we examined the catalytic activity of CYP106A1 from the Bacillus megaterium American Type Culture Collection 14581 strain. The CYP106A1 gene was cloned from B. megaterium, heterologously expressed in Escherichia coli, and purified. Potential electron partners and possible bacterial CYP106A1 substrates were identified by examining the oxidative activity toward a set of steroids in the presence of several reductase systems. The activities of CYP106A1 in a reconstituted system could not be achieved using rat NADPH-P450 reductase or a putidaredoxin reductase-putidaredoxin pair. However, the spinach redox proteins, a ferredoxin reductase-ferredoxin pair, were found to be efficient redox partners for CYP106A1. CYP106A1 catalyzes the hydroxylation of a set of steroids including testosterone, progesterone, 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and 11-deoxycortisol to produce monohydroxylated products as the major metabolites. These results suggest that CYP106A1 would be useful for the bioconversion of steroid hormones to hydroxylated products that can be used for industrial applications.

Comparison of the 17α-Hydroxylase/C17,20-Lyase Activities of Porcine, Guinea Pig and Bovine P450c17 Using Purified Recombinant Fusion Proteins Containing P450c17 Linked to NADPH-P450 Reductase

The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E. coli and purified for use in enzymatic assays to determine similarities and differences in 17α-hydroxylase and lyase activities. The fusion proteins were found to catalyze both the 17α-hydroxylation of progesterone (P4) and pregnenolone (P5) to 17α-hydroxylated P4 and P5 (17α-OH P4 and 17α-OH P5) followed by the C17,20-lyase reaction for the conversion of these C21-17α-hydroxylated steroids to C19-steroids (the C17,20-lyase reaction). These in vitro studies show that (a) porcine P450c17 possesses cytochrome b5 (b5)-stimulated C17,20-lyase activity that converts 17αOH-P4 to androstenedione (AD) but also converts 17α-OHP5 to dehydroepiandrosterone (DHEA); (b) guinea pig P450c17 possesses a b5-stimulated C17,20-lyase activity that converts 17α-OH P4 to AD but does not convert 17α-OH P5 to DHEA., and (c) bovine P450c17 possesses a b5-stimulated C17,20-lyase activity that converts 17α-OH P5 to DHEA but does not convert 17α-OH P4 to AD. Thus, the P450c17 of each species differs in its ability to catalyze in vitro the conversion of C21-steroids to C19-steroids. In addition, each P450c17 is capable of catalyzing additional hydroxylation reactions leading to low levels of 2α-, 6β-, 16- and 21-hydroxy-metabolites. Porcine P450c17 also catalyzes the b5-dependent synthesis of andien-β (androsta-5,16-dien-3β-ol) from P5.When the amino acid sequences of the three P450c17s were aligned there was an approximate 50% variation in the alignment identity (227 differences in the sequences of 509 amino acids). Alignment did not permit the assignment of specific amino acids or domains to the observed differences in enzymatic activities.

Purification of NADPH-P450 reductase (NPR) from Xenopus laevis and the developmental change in NPR expression

NADPH-P450 reductase (NPR) was purified from hepatic microsomes of Xenopus laevis. The electron transfer activity of purified NPR was 23.8 units/min/mg with horse cytochrome c. The aminopyrine demethylation activity of rat CYP2B1 with Xenopus NPR was 58.1 nmol/min/nmol. The corresponding cDNA was isolated from Xenopus liver. The homology in amino acid sequence deduced from NPR cDNA isolated from Xenopus liver was 80%, 78%, and 81% with human, rat, and rabbit NPR, respectively. Antibody against Xenopus NPR was prepared. The expression of NPR was investigated in various tissues and in early development by Western blotting. NPR was most abundantly expressed in the kidney, followed by the liver, lung, and heart. The brain had very low levels of NPR. The level of NPR protein was almost the same at all stages, 2-cell stage (st. 2), blastula (st. 8), gastrula (st. 12), tail bud (st. 26) and larva (st.35), examined in this study. We further investigated the distribution of NPR using whole-mount in situ hybridization. NPR mRNA was expressed in cement gland, lens placode, ear vesicle, mesencephalon, rhombencephalon, lymphatic vessel, and heart anlage in the embryo at stage 29. Xenopus NPR has similar properties to mouse and rat NPRs. Localization of NPR in Xenopus embryo was consistent with the abnormal region caused by NPR deficiency in mice.

Biochemical characterization of rat P450 2C11 fused to rat or bacterial NADPH-P450 reductase domains.

cDNAs coding for rat P450 2C11 fused to either a bacterial (the NADPH-cytochrome P450 BM3 reductase domain of P450 BM3) or a truncated form of rat NADPH-P450 reductases were expressed in Escherichia coli and characterized enzymatically. Measurements of NADPH cytochrome c reductase activity showed fusion-dependent increases in the rates of cytochrome c reduction by the bacterial or the mammalian flavoprotein (21 and 48%, respectively, of the rates observed with nonfused enzymes). Neither the bacterial flavoprotein nor the truncated rat reductase supported arachidonic acid metabolism by P450 2C11. In contrast, fusion of P450 2C11 to either reductase yielded proteins that metabolized arachidonic acid to products similar to those obtained with reconstituted systems containing P450 2C11 and native rat P450 reductase. Addition of a 10-fold molar excess of rat P450 reductase markedly increased the rates of metabolism by both fused and nonfused P450s 2C11. These increases occurred with preservation of the regioselectivity of arachidonic acid metabolism. The fusion-independent reduction of P450 2C11 by bacterial P450 BM3 reductase was shown by measurements of NADPH-dependent H(2)O(2) formation [73 +/- 10 and 10 +/- 1 nmol of H(2)O(2) formed min(-)(1) (nmol of P450)(-)(1) for the reconstituted and fused protein systems, respectively]. These studies demonstrate that (a) a self-sufficient, catalytically active arachidonate epoxygenase can be constructed by fusing P450 2C11 to mammalian or bacterial P450 reductases and (b) the P450 BM3 reductase interacts efficiently with mammalian P450 2C11 and catalyzes the reduction of the heme iron. However, fusion is required for metabolism and product formation.

Regulation ofCYP3A9Gene Expression by Estrogen and Catalytic Studies Using Cytochrome P450 3A9 Expressed inEscherichia coli

Sexual dimorphism in the expression ofCYP3A9,a novel form ofCYP3Afrom rat brain, is shown for the first time in rat brain as well as in rat liver.CYP3A9expression is female specific in rat liver as judged by its 10-fold higher expression in females than in males.CYP3A9gene expression was inducible by estrogen treatment both in male and in female rats. Ovariectomy of adult female rats elicited a drastic reduction on the mRNA level ofCYP3A9which could be fully restored by estrogen replacement. These results suggest that estrogen may play an important role in the female-specific expression of theCYP3A9gene. P450 3A9 recombinant protein was expressed inEscherichia coliby means of the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity with a specific content of 10.1 nmol P450/mg protein from solubilized fractions through two chromatographic steps. The purified P450 3A9 protein was active in the metabolism of imipramine, erythromycin, benzphetamine, and ethylmorphine as well as 17β-estradiol in a reconstituted system containing lipid and rat NADPH-P450 reductase. Of special interest is the finding that P450 3A9 can catalyze the formation of desipramine with a turnover number of 4.9 nmol/min/nmol P450, suggesting the possible involvement of this isoform in the metabolism of imipramine in brain. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture (1:1:1 mixture ofl-α-dilauroyl phosphatidylcholine,l-α-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH.

Expression, Purification, and Characterization of a Catalytically Active Human Cytochrome P450 1A2:Rat NADPH-Cytochrome P450 Reductase Fusion Protein

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression inEscherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5′ end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3′ end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser–Thr. This fusion plasmid was expressed inE. coliand the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP–agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochromecreduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufinO-deethylation and phenacetinO-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase inE. colienhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.

The Function of Recombinant Cytochrome P450s in Intact Escherichia coliCells: The 17α-Hydroxylation of Progesterone and Pregnenolone by P450c17

Studies are reported showing that recombinant P450c17, coexpressed with rat NADPH-P450 reductase or expressed as a fusion protein containing the domain of the P450 linked to the domain of NADPH-P450 reductase, function effectively in intact Escherichia colicells. Progesterone is rapidly hydroxylated by transformed E. colicells at rates as rapid as 50 nmol of steroid hydroxylated/min/nmol of P450 at 37°C. This rate measured in vivoequals or exceeds the best rates we have measured when reconstituting progesterone hydroxylase activity in vitrousing purified recombinant bovine P450c17 and purified recombinant rat NADPH-P450 reductase. The limits imposed in vivoby the availability of reducing equivalents (NADPH) and molecular oxygen are identified by showing the nearly fivefold increase in hydroxylation activity when glucose is present and the tendency for the constitutive respiratory activity of E. colito limit the availability of oxygen required for the P450-catalyzed reaction. The rate of progesterone metabolism is about 200 times faster by P450c17 coexpressed with NADPH-P450 reductase than when P450c17 functions with the constitutive electron transfer system of E. coli(flavodoxin and flavodoxin reductase). Expression of the fusion protein, termed rF450[mBov17A/mRatOR]L1, results in a rate of progesterone metabolism in vivoat 37°C of about 15 nmol of steroid hydroxylated/min/nmol of P450. Pregnenolone is actively metabolized to dehydroepiandrosterone at rates similar to those seen when the P450 activity is reconstituted in vitrowith cytochrome b 5. Experiments are described showing that the limited solubility of progesterone in water imposes a limit on the extent of steroid hydroxylated. The practicality of this type of P450-containing system for the bioconversion of large amounts of a chemical for the manufacture of speciality chemicals is discussed.

Characterization of a Fusion Protein between Human Cytochrome P450 1A1 and Rat NADPH-P450 Oxidoreductase inEscherichia coli

A cDNA of fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase was genetically engineered and expressed inEscherichia coliDH5α cells under the control of an inducibletacpromoter (Y. J. Chun, T. Shimada, and F. P. Guengerich, (1996)Arch. Biochem. Biophys.330, 48-58).E. colimembranes of transformed cells showed much higher P450 1A1-dependent monooxygenase and NADPH-P450 reductase activities than pCW control vector or P450 1A1 expression vector-transformed cells. EthoxyresorufinO-deethylase and methoxyresorufinO-demethylase were 22-fold and 11-fold higher than the control activity, respectively. α-Naphthoflavone and β-naphthoflavone strongly inhibited P450 1A1 activity of the fusion protein, with α-naphthoflavone being more potent than β-naphthoflavone. Divalent cations (e.g. Ca2+and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reductase activity. These results demonstrate that this fusion protein inE. colimembrane may be a useful model for elucidating details of protein-protein interactions between P450 and NADPH-P450 reductase in the endoplasmic reticulum of mammalian cells.

Baculovirus Expression and Purification of Human and Rat Cytochrome P450 2E1

High-level expression of human and rat cytochrome P450 2E1 (CYP2E1) was achieved using a baculovirus expression system. A full length cDNA encoding human CYP2E1 was cloned from a human liver cDNA library and sequenced using the dideoxy sequencing method. Insect cells were infected with the homologous recombinant baculoviruses containing the human and rat CYP2E1 cDNAs, respectively. The infected cells were harvested at a time when 450-nm peak intensities were at a maximal level and there was no 420-nm peak observed in the reduced CO difference spectrum. Both human and rat CYP2E1 were then purified to electrophoretic homogeneity by a relatively rapid and efficient procedure. The specific contents of the purified human and rat CYP2E1 were 13.8 and 17.0 nmol/mg protein, respectively. The λmaxof the reduced CO difference spectra of both purified rat and human CYP2E1 was found to be 451.5 nm. When the purified rat and human CYP2E1 were reconstituted with purified rat NADPH–P450 reductase and human cytochrome b5, they were able to metabolize several known CYP2E1 substrates: chlorzoxazone,p-nitrophenol, acet- aminophen, and carbon tetrachloride. Interestingly, cytochrome b5markedly stimulated the CYP2E1-mediated two-electron oxidation of the first three substrates, while it had almost no effect on the presumed one-electron reduction of carbon tetrachloride.

Construction of a Human Cytochrome P450 1A1:Rat NADPH–Cytochrome P450 Reductase Fusion Protein cDNA and Expression inEscherichia coli,Purification, and Catalytic Properties of the Enzyme in Bacterial Cells and after Purification

A plasmid (pCW) was modified to code for a fusion protein consisting of the complete sequence of human cytochrome P450 (P450) 1A1 (with only the second amino acid changed) in the N-terminal portion connected by a Ser-Thr linker to the portion of rat NADPH–P450 reductase beginning at amino acid 57. This plasmid was used to express the fusion protein inEscherichia coliDH5α cells and the protein was purified from detergent-solubilized bacterial membranes using DEAE and 2′,5′-ADP agarose chromatography. The purified fusion protein catalyzed benzo[a]pyrene 3-hydroxylation, 7-ethoxyresorufin O-deethylation, and zoxazolamine 6-hydroxylation. Catalytic activity was not increased in the presence of added NADPH–P450 reductase, cytochromeb5, or phospholipid. The fusion protein could also transfer electrons to cytochromescandb5but not P450 1A2. The same oxidation products of benzo[a]pyrene were formed with the purified fusion protein and the fusion protein functioning in bacterial cells. The catalytic activity of the human P450 1A1 fusion protein toward several substrates is markedly less than that of a similar fusion protein constructed with rat P450 1A1, in line with the reported differences in catalytic activities of the rat and human P450 1A1 enzymes. The purified fusion protein also oxidized (+)- and (−)-benzo[a]pyrene 7,8-dihydrodiols and eight aryl and heterocyclic amines to genotoxic products, in the absence of added NADPH–P450 reductase. The demonstration of catalytic activities of the human fusion protein within bacterial cells suggests the prospect of utilizing such cellular systems for production of human P450 metabolites.

1121 Differential expression of TAG-1/F3 and L1 AxCAM subgroup members in the developing and adult rat nervous system

An enzymatically active human cytochrome P450 (P450) 1A2:rat NADPH-P450 reductase fusion protein was purified and partially characterized following heterologous expression inEscherichia coli. A cDNA was engineered to include the coding sequence for human P450 1A2 at its 5′ end (up to but not including the stop codon) fused in-frame to the coding sequence for a truncated (soluble) rat NADPH-P450 reductase at its 3′ end via an oligonucleotide sequence encoding the hydrophilic dipeptide Ser–Thr. This fusion plasmid was expressed inE. coliand the recombinant protein was purified from the detergent-solubilized membrane fraction via sequential DEAE, ADP–agarose, and hydroxylapatite chromatographies. The purified protein has the spectral characteristics of human P450 1A2 and cytochromecreduction activity comparable to rabbit NADPH-P450 reductase. The fusion protein catalyzed 7-ethoxyresorufinO-deethylation and phenacetinO-deethylation to appreciable levels in the presence of NADPH and phospholipid. While these activities were comparable to those of other such P450:NADPH-P450 reductase fusion proteins, they were lower than those of the system reconstituted from its individual hemoprotein and flavoprotein components. Nevertheless, the production of a functional, catalytically self-sufficient monooxygenase inE. colienhances the prospect of using bacterial systems for production and characterization of human P450 drug metabolites as well as for biodegradation of chemicals in the environment.

The High-Level Expression in Escherichia coli of the Membrane-Bound Form of Human and Rat Cytochrome b 5 and Studies on Their Mechanism of Function

A T7 expression system is described for the high-level production in Escherichia coli of the membrane-bound form of human and rat cytochrome b 5. The cDNAs of b 5 have been engineered to contain a coding sequence for a four-member histidine domain at the amino-terminus of the recombinant protein permitting the use of a nickel-chelate affinity column for rapid purification of the detergent-solubilized hemoprotein. Results are presented demonstrating the ability of the purified recombinant b 5 proteins to stimulate the rate of oxidation of 17α-hydroxypregnenolone to dehydroepiandrosterone, catalyzed by bovine P450 17A, and to stimulate the 6β-hydroxylation of testosterone, catalyzed by human P450 3A4. These P450-catalyzed reactions have been used to compare the properties of different forms of b 5. Purified b 5 can serve as a "coupling protein" as illustrated by its inhibition of NADPH oxidation, catalyzed by a fusion protein containing the heme domain of P450 3A4 linked to rat NADPH-P450 reductase, and the associated inhibition of hydrogen peroxide formation. Kinetic studies show the formation of a complex of the flavoprotein, NADPH-P450 reductase, with b 5 for the rapid transfer of electrons from NADPH.

Purification and Enzymatic Properties of a Recombinant Fusion Protein Expressed in Escherichia coli Containing the Domains of Bovine P450 17A and Rat NADPH-P450 Reductase

A fusion protein containing the heme domain of bovine cytochrome P450 17A and the flavin domains of rat NADPH-cytochrome P450 reductase has been genetically engineered by linking the modified cDNAs for each gene with the codons for serine and threonine. Transformation of Escherichia coli (DH5α) and growth under defined conditions permits expression of 600-700 nmol of membrane-bound fusion protein per liter of growth medium (approximately 4% of cellular protein). A method has been developed for the solubilization, isolation, and purification to homogeneity of this protein. In the presence of NADPH the purified fusion protein catalyzes the 17α-hydroxylation of progesterone and pregnenolone as well as the conversion of 17α-hydroxypregnenolone to dehydroepiandrosterone. The 17,20-lyase activity is enhanced sixfold by the addition of purified rat liver cytochrome b5. Further, dehydroepiandrosterone is slowly metabolized to a number of additional more polar metabolites while 17α-hydroxy-pro-gesterone is slowly converted to dihydroxy-progesterone metabolites as well as a small amount of androstenedione in a reaction not influenced by cytochrome b5. Use of 5α-pregnan steroids as substrates show the importance of the 3β-hydroxyl group for cytochrome b5 stimulated 17,20-lyase activity. Studies investigating the factors affecting electron transport between the flavin and heme domains suggest that the protein exists as a tight complex functioning as a self-contained biocatalytic unit.

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.