Cytochrome P450c17-expressing Escherichia coli as a first-step screening system for 17alpha-hydroxylase-C17,20-lyase inhibitors

We have designed and synthesized a number of cytochrome P450 17alpha-hydroxylase-C17,20-lyase (P450c17) inhibitors with the aim of inhibiting androgen synthesis. To select the most potent inhibitors, we initially used human testicular microsomes, which have a high level of expression of this enzyme. However, due to lack of availability of human tissue and variability among the samples, we utilized recombinant human enzyme expressed in Escherichia coli. We designed a simple and economical protocol based on the report that recombinant bovine P450c17 can be functionally active in live bacteria. In the assay we report here, we substituted high-performance liquid chromatography product isolation with a rapid biochemical acetic acid releasing assay and utilized intact P450c17-expressing E. coli for the source of the enzyme. Enzymatic parameters of the bacterial system (Km = 5.1 x 10(-7) M, Vmax = 15.0 pmol/min/mg) were similar to those of human testicular microsomes (Km = 4.8 x 10(-7) M, Vmax = 40.0 pmol/min/mg), and our compounds displayed a similar pattern of inhibition in both systems. This new system is a fast, reliable, and reproducible method for screening P450c17 inhibitors. Furthermore, it eliminates our dependence on human tissue and potential data fluctuations caused by variations in enzymatic activity between donors.     

Evidence for requirement of NADPH-cytochrome P450 oxidoreductase in the microsomal NADPH-sterol Delta7-reductase system

Rabbit antibodies raised against the hydrophilic part of microsomal NADPH-cytochrome P450 oxidoreductase (denoted fpT) demonstrated a marked ability to inhibit NADPH-sterol Delta7-reductase activity. In addition, trypsin and proteinase K treatment of microsomes removed almost all microsomal electron transfer constituents from the microsomes, but the Delta7-reductase activity could be reconstituted by adding detergent-solubilized NADPH-cytochrome P450 oxidoreductase (denoted OR). Furthermore, after solubilization from microsomes, the Delta7-reductase activity could be reconstituted with OR in a DEAE-cellulose column chromatography eluate fraction, which contained little OR activity. In the microsomal system, carbon monoxide, ketoconazole, and miconazole, specific inhibitors of cytochrome P450, had no effect on Delta7-reductase activity. These results provide the first evidence of an essential requirement of OR, which is distinct from cytochrome P450, in the NADPH-sterol Delta7-reductase system. EDTA, o-phenanthroline and KCN markedly lowered Delta7-reductase activity in a dose-dependent manner. Among metal ions tested, only ferric ion restored the reductase activity in the EDTA-treated microsomes. These results sugguest that NADPH-sterol Delta7-reductase is membrane-bound iron-dependent protein embedded in the microsomal lipid bilayer.     

Inhibition of cytochrome P450 mediated enzyme activity by alkylphosphocholines

The inhibitory potency of four alkylphospholipids: rac-1-O-phosphocholine-2-hydroxy-octadecane (rac-2-OH), rac-1-O-phosphocholine-2-O-acetyl-octadecane (rac-2-O-acetyl), rac-1-O-phosphocholine-2-amino-octadecane (rac-2-NH2) and rac-1-O-phosphocholine-2-N-acetyloctadecane (rac-2-N-acetyl), on the cytochrome P450-dependent monooxygenase activity has been evaluated. The IC50 values of the alkylphosphocholines with 7-ethoxycoumarin as substrate in liver microsomal fractions of PB-treated rats and with a reconstituted CYP2B1: NADPH-P450-reductase system are in the range of 3.2-5.0 microM and 2.8-3.5 microM, respectively. Lineweaver-Burk plots with the inhibitors in concentrations that were found to cause roughly a 50% inhibition and with 7-ethoxycoumarin as substrate revealed for all four alkylphospholipids a competitive inhibition type. The degree of the competitive inhibition is quantified by the Ki values. With liver microsomal fractions of PB-treated rats, the Ki values of rac-2-OH (Ki = 1.36 microM) and rac-2-O-acetyl (Ki = 1.33 microM) differs slightly from those of rac-2-NH2 (Ki = 2.2 microM) and rac-2-N-acetyl (Ki = 2.2 microM), but with the reconstituted CYP2B1: NADPH-P450-reductase system all Ki values are in the small range of 1.8 - 2.6 microM, indicating that the short substituted group at the 2-position (OH; O-acetyl; NH2; N-acetyl) of the long chain octadecanol part of the phosphodiesters exhibit no essential role on the strong inhibitory potency of these alkylphosphocholines on the 7-ethoxycoumarin-O-deethylase activity.     

Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.     

The regulation of ovine placental steroid 17 alpha-hydroxylase and aromatase by glucocorticoid

Parturition in the pregnant sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to estrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha), steroid C-17,20-lyase, and possibly aromatase. We have investigated the activity levels of aromatase and 17 alpha-hydroxylase in placental microsomes in late pregnancy and dexamethasone-induced labor. Over the gestational period of 118-140 days basal levels of placental aromatase were relatively constant [mean value (+/- SD) of 5.6 +/- 1.6 pmol min-1 mg microsomal protein-1 (n = 10)]. Steroid 17 alpha-hydroxylase activity was undetectable [less than 0.5 pmol min-1 mg microsomal protein-1 (n = 7)]. In six animals in labor induced with infusion of dexamethasone into the fetus, placental aromatase activity had a mean value of 14.0 +/- 2.5 pmol min-1 mg protein-1; placental steroid 17 alpha-hydroxylase, measured in four of the animals, had a mean (+/- SD) activity of 319 +/- 58 pmol min-1 mg microsomal protein-1. Immunoblotting of placental microsomal preparations with specific antibodies to cytochrome P-450(17)alpha and NADPH-cytochrome P-450-reductase indicated that the glucocorticoid-induced activity of 17 alpha-hydroxylase was associated with increased content of cytochrome P-450(17)alpha. Northern blotting with a cDNA probe for cytochrome P-450(17)alpha showed that glucocorticoid increased the levels of mRNA for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of steroid biosynthesis modifiers on progesterone biotransformation by recombinant yeasts expressing cytochrome P450cl7

Using recombinant microorganisms S. cerevisiae GRF18/YEp 5117α, expressing bovine adrenocortical cytochrome P450cl7, we have studied the effect of various modifiers of steroid biosynthesis on the relationship between reactions of the 17α-hydroxylation and 20α-reduction of progesterone. Dexamethasone and metyrapone had no effect on the reaction of progesterone 17α-hydroxylation and 20α-reduction of 17α-hydroxyprogesterone. Mifepriston and danazol did not covalently modify amino acid residues of the cytochrome P450cl7 or its heme group under the conditions of progesterone biotransformation by recombinant yeasts. Ketokonazole, mifepriston and danazol were found to be low-affinity competitive inhibitors, but the 20-dihydroderivatives of progesterone were mixed type inhibitors of the cytochrome P450cl7. All modifiers used did not affect the functional properties of the yeast analog of 20α-hydroxysteroid dehydrogenase. Based on the effect on catalytic parameters of the cytochrome P450cl7, the all modifiers used can be arranged in the following order: 20β-dihydroprogesterone (maximal effect) > mifepriston = ketokonazole > 20α-dihydroprogesterone > danazol > dexamethasone, metyrapone (without effect).     

Catalytic properties of the human cytochrome P450 2E1 produced by cDNA expression in mammalian cells.

A full-length cDNA encoding human cytochrome P450 2E1 was expressed in mammalian cell lines using the vaccinia virus expression system. Immunoblot analysis showed that the expressed protein reacted with a polyclonal antibody against rat 2E1 and comigrated with P450 2E1 from human liver microsomes. P450 2E1 expressed in Hep G2 cells, a human cell line which contains both cytochrome b5 and NADPH:P450 oxidoreductase, was able to metabolize several known P450 2E1 substrates: N-nitrosodimethylamine (NDMA), N-nitrosomethylbenzylamine (NMBzA), p-nitrophenol, phenol, and acetaminophen. Apparent Km and Vmax values for NDMA demethylation were 22 microM and 173 pmol/min/mg microsomal protein, respectively. P450 2E1 expressed in TK-143 cells, which do not contain b5, displayed Km and Vmax values of 31 microM and 34 pmol/min/mg microsomal protein, respectively. Incorporation of purified rat liver b5 into TK-143 microsomes increased the Vmax 2.2-fold and decreased the Km to 22 microM. Addition of b5 to Hep G2 microsomes resulted in a 1.6-fold increase in Vmax, but showed no effect on the Km. P450 2E1 expressed in Hep G2 cells was shown to metabolize NMBzA with a Km of 47 microM and Vmax of 213 pmol/min/mg microsomal protein. Addition of b5 lowered the Km to 27 microM, but had no effect on Vmax. These results demonstrate conclusively that P450 2E1 is responsible for the low Km forms of NDMA demethylase and NMBzA debenzylase observed in liver microsomes and that these activities are affected by cytochrome b5.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17-hydroxyprogesterone, androstenedione and testosterone. The transition from C₂₁ to C₁₉ steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at the steady state for both reactions using [1,2-³H]progesterone and [1,2-³H]17-hydroxyprogesterone as substrates. In the testis and male and female duodena, the K_m values for progesterone 17-hydroxylation were 14.2, 23.8 and 23.2 nM, whereas the V_max values were 105, 3.5 and 3.1 pmol/mg protein/min, respectively. With respect to C-17,20 lyase activity, the K_m values for exogenous 17-hydroxyprogesterone were 525, 675 and 637 nM, whereas the V_max values were 283, 7.8 and 7.8 pmol/mg protein/min, respectively. However, when the K_m values were calculated with respect to intermediate 17-hydroxyprogesterone formed from progesterone, they were similar to the K_m values for 17-hydroxylase, being 15, 31.4 and 24.8 nM, whereas the V_max values were 26.3, 2 and 1.8 pmol/mg protein/min, respectively. The similarity of K_m values is due to the fact that the relative androgen formation efficiency (bond scission events/total 17-hydroxylation events ratio) was remarkably constant in both testicular and duodenal incubates, irrespective of progesterone concentration. Efficiency values were 2-fold higher in duodenal tissue (0.54) than in testis (0.25). Estradiol-17β inhibited 17-hydroxylation but not bond scission on intermediate 17-hydroxyprogesterone, because it did not affect the efficiency value. Rat duodenal P450c17 has the same substrate affinity, a lower specific activity and a higher androgen formation efficiency than testicular P450c17.     

Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae.

The most common cause of congenital adrenal hyperplasia is deficiency of cytochrome P450c21 (21-hydroxylase), which catalyzes the synthesis of adrenal steroids. We have cloned the human P450c21 cDNA into yeast expression vectors under the control of either the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) promoter or the aldehyde-dehydrogenase (ADH) promoter. P450c21 RNA, protein, and enzyme activity can be detected, indicating that both promoters drive the synthesis of P450c21. The expressed P450c21 catalyzes the conversion of both of its substrates, with Km and Vmax values of 0.33 microM and 280 nmoles/hr.nmole of P450c21 protein for progesterone, and 0.23 microM and 450 nmoles/hr.nmole for 17-hydroxyprogesterone. These kinetic properties are similar to those of human P450c21 expressed in COS-1 cells. The microsomal fraction containing P450c21 exhibited an absorption peak at 450 nm upon binding to CO, demonstrating its hemoprotein nature. The CO-difference spectra indicated that there were about 0.08 nmole P450c21 hemoprotein/mg microsomal protein. Coupling this expression system with site-directed mutagenesis, the Asn-172 mutant of P450c21 had about 20-100 lower Vmax values; yet it retained normal affinity toward both substrates. This mutant protein also exhibited an altered absorbance with a peak at 420 nm rather than at 450 nm.     

Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.     

Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius

Summary Microsomal and soluble fractions of Pleurotus pulmonarius exhibited a reduced carbon monoxide difference spectrum with P450 maxima at 448nm and 450–452nm respectively. Substrate induced Type I spectra were observed on addition of benzo(a)pyrene to both fractions. Benzo(a)pyrene hydroxylation was measured using the aryl hydrocarbon hydroxylase assay and was observed to be P450 dependent as indicated by carbon monoxide inhibition together with the substrate binding characteristics. The activity of the fractions were observed to give K_m of 200mM and 660mM and V_max of 1.25 nmol/min/nmol P450 and 0.57 nmol/min/nmol P450 for the microsomal and cytosolic fractions respectively.     

Increased expression of cytochrome P450 IIIA2 in male rat liver after dietary vitamin A supplementation

In this study dietary vitamin A supplementation (25 IU/g diet) was assessed for its effect on hepatic microsomal P450 content and on P450 enzyme-specific drug oxidase activities in rats. Intake of the supplemented diet by male rats over a 15-week period resulted in a fivefold increase in hepatic vitamin A stores over those measured in control liver from rats that received a balanced diet without vitamin A supplementation. Serum retinol was unchanged and there was no evidence of hepatocellular injury in any of the animals. There was a 26% increase in P450 content in vitamin A-supplemented rat liver and regioselective androst-4-ene-3,17-dione (androstenedione) and progesterone hydroxylation revealed changes in several P450 pathways. Thus, androstenedione 16 alpha-hydroxylation (P450 IIC11-mediated) and progesterone 21-hydroxylation (P450 IIC6-mediated) were decreased slightly to 80 and 74% of respective control activities while P450 IIA1/2-dependent androstenedione 7 alpha-hydroxylation was slightly increased. In contrast, the 6 beta-hydroxylations of androstenedione and progesterone were increased to 169 and 152% of control following dietary supplementation. Kinetic analysis of androstenedione 6 beta-hydroxylation revealed an increase in maximal reaction velocity (Vmax 4.00 +/- 0.47 vs 2.20 +/- 0.10 nmol/min/mg protein) but the Km was unchanged, suggesting an increase in enzyme concentration. Consistent with this assertion, immunoquantitation of the steroid 6 beta-hydroxylase, P450 IIIA2, revealed a 158% increase in the microsomal expression of this enzyme (9.8 +/- 2.7 vs 6.2 +/- 1.3 ng/micrograms microsomal protein). From these studies it now seems clear that vitamin A, as a dietary additive in nontoxic doses, has the capacity to alter the activity of hepatic microsomal drug oxidases by modulating the expression of P450 enzymes.     

Occurrence and inducibility of cytochrome P450IIIA in maternal and fetal rats during prenatal development

The purpose of this study was to quantify cytochrome P450IIIA1 in fetal and maternal livers of uninduced and pregnenolone-16 alpha-carbonitrile (PCN) induced rats during the course of prenatal development. The activities and levels of P450IIIA in hepatic microsomes from maternal rats and fetuses at 15-21 days of gestation were measured by triacetyloleandomycin (TAO) inhibited debenzylation of (benzyloxy)phenoxazone and by immunoassay with defined antiserum specific for P450IIIA. P450IIIA was not detectable (less than 10 pmol/mg for maternal microsomes and less than 2 pmol/mg for fetal microsomes) by immunoassay in uninduced maternal or fetal livers. In hepatic microsomes from PCN-induced dams, values ranged from 59.3 to 116 micrograms P450IIIA1/mg of protein during the same gestational period. Changes in debenzylase activity of 15.9-46.5 pmol of resorufin (mg of protein)-1 min-1 were consistent with these findings as were the changes in TAO-inhibitable debenzylase activity. In the transplancentally induced fetal liver, debenzylase activity increased steadily from 0.19 pmol of resorufin mg-1 min-1 at day 15 to 9.34 pmol of resorufin mg-1 min-1 at day 21 and was paralleled by the TAO-inhibitable activity that ranged from 0.09 pmol of resorufin mg-1 min-1 at day 15 to 3.33 pmol of resorufin mg-1 min-1 at day 21. The amount of immunoreactive P450IIIA1 also increased from 0.5 to 28.7 micrograms/mg of microsomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

绿盲蝽P450基因的克隆及增效醚对P450酶活性的抑制作用

为探讨P450介导的绿盲蝽Apolygus lucorum(Meyer-Dür)抗药性机制,合理使用杀虫药剂,本研究通过活体和离体抑制实验发现,增效醚(PBO)对绿盲蝽P450酶活性有显著的抑制作用:在处理时长为24h时,P450酶活性由未处理时的12.02pmol/min/mgPro.下降至1.63pmol/min/mgPro.,PBO对P450酶的抑制中浓度为0.256mmol/L。生物测定结果表明,PBO对三氟氯氰菊酯具有显著增效作用,增效7.2倍,而对吡虫啉、灭多威、马拉硫磷无显著增效作用。利用RT-PCR及RACE技术对绿盲蝽P450基因进行克隆,获得了2条CYP4家族基因,全长均为1631bp,含有完整的开放阅读框,编码501个氨基酸;序列比对表明这是一对等位基因,含有CYP4家族所有保守特征序列;同源性比较及系统发育分析显示这2个基因编码的氨基酸序列与褐飞虱Nilaparvata lugens CYP4CE1亲缘关系最近,同源性分别为41.5%和41.1%。     

Selective potent restriction of P450b- but not P450e-dependent 7,12-dimethylbenz[a]anthracene metabolism by the microsomal environment

The prototypic members of the rat liver cytochrome P450IIB subfamily, P450b and P450e, differ by only 13 amino acids and yet purified P450b is considerably more active than P450e for all known substrates. A unique regioselectivity difference between cytochromes P450b and P450e for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and a genetic deficiency in P450e expression in the Marshall (M520/N) rat strain have been exploited to determine the microsomal contributions of the respective forms toward the metabolism of DMBA. The total contribution to metabolism by each isozyme has been assessed based on the sensitivity to rabbit anti-P450b/e IgG and comparison with microsomal P450b and P450e content as measured by Western blots. Liver microsomes from untreated M520/N rats do not express detectable levels of P450e but express P450b at a level that is 2-fold higher than that of P450e in liver microsomes from untreated F344 rats (50 pmol/mg). However, only 4% of the constitutive DMBA metabolizing activity of liver microsomes from the M520/N rat strain could be inhibited by anti-P450b/e IgG. A 30-fold induction of hepatic P450b by phenobarbital (PB) was also completely ineffective in increasing P450b-dependent DMBA metabolism. PB treatment had no appreciable effect on either the levels of expression of P450b protein or P450b-dependent DMBA metabolism, in M520/N lung and adrenal microsomes. In contrast, PB treatment of F344 rats considerably increased P450b/e-dependent metabolism by liver, lung, and adrenal microsomes. The regioselectivity of the anti-P450b/e-sensitive metabolism (predominantly 12-methyl hydroxylation), however, indicated a much greater contribution from P450e than P450b in every tissue examined despite a several fold higher expression of P450b than of P450e. P450b was expressed constitutively in lung microsomes from both strains but again failed to exhibit appreciable DMBA metabolizing activity. Based on these activities and microsomal P450b contents, P450b consistently exhibited turnover numbers (0.02-0.15 nmol/nmol P450b/min) that were at least 10-fold lower than those of pure P450b. In contrast, the calculated turnover numbers for microsomal P450e were consistently comparable to those of pure P450e (approximately 1 nmol/nmol P450e/min).     

Comparison of the hamster and human adrenal P450c17 (17 alpha-hydroxylase/17,20-lyase) using site-directed mutagenesis and molecular modeling

In order to understand the activity specificity of the hamster cytochrome P450 17 alpha-hydroxylase/17,20-lyase (P450c17), we have studied its structure/activity using three hamster P450c17 recombinant mutants (T202N/D240N/D407H). In transiently transfected COS-1 cells, the mutation T202N reduced 17 alpha-hydroxylation of pregnenolone and progesterone to 24 and 44% of wild type (WT), respectively, followed by reduced 17,20-cleavage to 71 and 67%, respectively. On the other hand, the mutation D240N decreased specifically 17,20-lyase activity to 61% of WT when incubated with pregnenolone while the mutation D407H only decreased 17 alpha-hydroxylation to 46% when incubated with progesterone.To comprehend the altered activity profiles of these hamster P450c17 mutants, we have elaborated a 3D model of the hamster P450c17 and compared it to our preceding model of the human P450c17. Analysis of the mutants with this model showed that, without direct contact to the substrates, these mutations transmit structural changes to the active site. By analogy, these results support the concept that any cellular changes modifying the external structure of P450c17, such as phosphorylation, could have influence on its active site and enzymatic activities.     

Synthesis of hydroxy derivatives of highly potent non-steroidal CYP 17 inhibitors as potential metabolites and evaluation of their activity by a non cellular assay using recombinant human enzyme.

Inhibition of CYP 17 is a promising strategy for the treatment of prostate cancer. Recently two non-steroidal compounds with high in vitro activity were synthesized in our group (BW19 and BW95). However, after a few hours they showed in vivo a strong decrease in their activity. This might be due to a fast biodegradation. Potential hydroxy and epoxy metabolites were synthesized and their inhibitory activities were tested by a new non-cellular assay using recombinant enzyme. As source, membrane fractions of E. coli pJL17/OR coexpressing human CYP 17 and rat NADPH-P450-reductase were, used. Showing a high and constant CYP 17 activity and a fast and easy isolation procedure the new method was advantageous compared with the microsomal assay. Interestingly, all the new synthesized hydroxy and epoxy compounds except one showed a lower inhibition of CYP 17 than the parent compounds. Thus, the loss of in vivo activity may be partly explained.     

Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.     

Cytochrome P450 and steroid 21-hydroxylation in microsomes from beef adrenal cortex

Cytochrome P450 in beef adrenal cortex microsomal preparations reacted with progesterone and with 17-hydroxyprogesterone at pH 7.4 to produce Type I spectral changes. The magnitude of the spectral shift produced by addition of progesterone or 17-hydroxyprogesterone was related to the concentration of cytochrome P450 (over P450 concentration range of 0.1 to 0.3 μM). Prior saturation of cytochrome P450 with 17-hydroxyprogesterone prevented further spectral shift with the addition of progesterone. On the other hand, saturation of cytochrome P450 with progesterone decreases the expected shift with 17-hydroxyprogesterone by more than 50% but did not prevent the shift. The difference spectra were diminished by more than 50% at pH 9.0.The addition of NADPH resulted in loss of the spectral shifts and production of 21-hydroxylated products, predominantly DOC and 11-deoxycortisol. These reactions were not inhibited by their specific products. The rate of 21-hydroxylation was linearly related to microsomal protein (and microsomal P450) concentration. The 21-hydroxylation of progesterone was competitively inhibited by 17-hydroxyprogesterone; inhibition of the 21-hydroxylation of 17-hydroxyprogesterone by progesterone was not demonstrated.