Steroid structure and function V. A-ring conformation in 17-hydroxy-6 alpha-methylprogesterone

The molecular conformation of 17-hydroxy-6 alpha-methylprogesterone has been determined crystallographically and is compared with 17-hydroxy-progesterone, 17-acetoxyprogesterone and 17-acetoxy-6 alpha-methylprogesterone (MPA). The analysis demonstrates that the 6 alpha-methyl substituent is not sufficient by itself to induce inversion of the A-ring. Consequently, the inverted form observed in MPA and proposed to be responsible for high affinity binding to the progesterone receptor appears to be induced by the combined long range influence of 17 alpha-acetoxy substituent and the direct interaction of the 6 alpha-methyl group with the flexible A-ring.     

Synthesis and biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate

The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3,2 0-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches.     

Glucocorticoid properties of progestogens

Single oral doses of progesterone, 17-hydroxyprogesterone acetate, medroxyprogesterone acetate, megestrol acetate, cortisol, or placebo were given to groups of fasting young men. Eosinophils, plasma glucose and corticosteroids were determined at hourly intervals for 8 hours. Maximum responses were compared between groups receiving different treatments. Progesterone is free of glucocorticoid properties in these tests, but medroxyprogesterone acetate has about 50% and megestrol acetate about 25% the eosinopenic and hyperglycemic activity of an equal weight of cortisol in the dose range tested. 17-hydroxyprogesterone acetate has no eosinopenic or hyperglycemic activity, but suppresses plasma corticosteroids similarly to equal weights of medroxyprogesterone acetate or megestrol acetate.     

Steroid structure and function:I. Conformational transmission in 17alpha-acetoxy progesterone

The molecular conformation of 17alpha-acetoxy progesterone has been determined crystallographically and is compared with that of progesterone. The 17alpha-acetate substituent restricts the flexibility of the progesterone side chain, strains bond lengths in the C- and D-rings, and has long range effects on the A-ring conformation. The A-ring adopts a perfect sofa conformation similar to that observed in one conformational isomer of progesterone. Consequently this progesterone isomer is proposed to be that best suited to binding in the rabbit and human uterus.     

Affinities of progestogen and estrogen receptors in rabbit uterus for synthetic progestogens

The affinities of progestogen and estrogen receptors of rabbit uterus for a number of synthetic progestogens in clinical use and some analogues have been measured. Progesterone, 17-hydroxyprogesterone caproate, the chlormadinone, megestrol and cyproterone acetates had similar affinities for the progestogen receptor. Medroxyprogesterone acetate and particularly DU-41164 possessed much higher affinities. 17-hydroxyprogesterone or other agents with a free 17-hydroxyl had much lower receptor affinities than the corresponding 17-esters.Of the nortestosterone derivatives tested, norethisterone equalled progesterone in affinity for the progestogen receptor while d-norgestrel and Wy-4355 were more active. Norethynodrel and ethynodiol diacetate had much lower receptor affinities than progesterone. These results are discussed in relation to possible metabolic bioactivation.Only norethynodrel and ethynodiol (free alcohol) showed marked affinity for the estrogen receptor.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Melengestrol acetate and megestrol acetate are prostatic tumor inhibiting agents

We had previously reported that 6-methylene progesterone, an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone to dihydrotestosterone, markedly inhibited growth of the androgen-dependent Dunning R3327-H rat prostatic tumors. We now find that the progesterone derivatives melengestrol acetate (MGA) and megestrol acetate (MA) inhibit both the androgen-dependent (Dunning R3327-H) and the androgen-independent (Dunning R3327-AT3) prostatic tumors. Growth of the AT3 tumors was suppressed by approximately 53% after 9 days of daily s.c. injections with MGA at 10 mg/kg body weight. MGA also caused a 54% weight reduction of the ventral prostate and a 53% reduction of the seminal vesicles. Adrenal weights were reduced by 42%. A 24-day oral treatment with MGA (at approximately 15-17 mg/(kg.day)) inhibited AT3 tumor growth by 59% and caused a weight reduction in the following tissues: prostate (46%), seminal vesicles (19%), testes (12%), and adrenals (52%). Under the same protocol, MA inhibited AT3 tumor growth by 32% and reduced the weight of the ventral prostate by 49% and the weight of the adrenals by 18%, but had no effect on the seminal vesicles and testes. The extent of the MGA-induced prostatic regression was accompanied by cytological changes similar to those effected by 6-methylene progesterone, i.e., shrinking of the acinar epithelium. The AT3 tumors in MGA-treated rats displayed a limited degree of apoptosis. Atrophy of the adrenal cortex and lowered plasma levels of corticosterone and dihydroepiandrosterone were also observed. A therapeutic role for MGA and MA against androgen-independent prostatic neoplasms in man is forecast by these observations.     

Megestrol acetate drives endometrial carcinoma cell senescence via interacting with progesterone receptor B/FOXO1 axis

Megestrol acetate is a common and efficient anticancer progesterone. To explore the activity and the therapeutic mechanisms of megestrol acetate in endometrial cancer, human endometrial cancer cell lines Ishikawa and HHUA overexpressing progesterone receptor A (PR-A) and progesterone receptor B (PR-B) were treated with megestrol acetate. Cell viability, apoptosis, cycle arrest, and senescence, as well as the expressions of p21 and p16, two hallmarks of cellular senescence, were evaluated. Compared with the control, >10 nmol/L megestrol acetate treatment could significantly reduce endometrial cancer cell growth, and induce the irreversible G1 arrest and cell senescence. The expression of cyclin D1 in megestrol acetate treated cells was downregulated, while the expressions of p21 and p16 were upregulated via PR-B isoform. FOXO1 inhibitor AS1842856 could significantly abrogate megestrol acetate-induced cell senescence, suggesting that FOXO1 was involved in megestrol acetate/PR-B axis. These findings may provide a new understanding for the treatment of human endometrial cancer.     

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.     

Interaction of [3H]nomegestrol acetate with cytosolic progesterone receptors from the rat uterus

The binding characteristics of the progestin 17 alpha-acetoxy-6-methyl-19-[3H]norpregna-4,6-diene-3,20-dione, nomegestrol acetate ([3H]NOM-Ac) to progesterone receptors (PgRs) of uterus were determined in the rat. Scatchard plot analysis of the equilibrium binding data showed that [3H]NOM-Ac binds to uterine PgR with a Kd of 5.44 +/- 1.27 nM and a Bmax of 1.51 +/- 0.11 pmol/mg protein. Analysis of dissociation kinetics showed that [3H]NOM-Ac dissociates slowly from the PgR, k - 1 = 4.9 +/- 0.5 10(-5) s-1. Competition experiments against [3H]NOM-Ac showed the specificity of the binding with a sequence in relative affinity as follows: ORG 2058 greater than P greater than NOM-Ac greater than medroxyprogesterone acetate greater than megestrol acetate greater than cyproterone acetone greater than NOM.     

Use of a new radioactive ligand, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H]19-nortestosterone for the estimation of androgen receptors in rat liver cytosol

The use of the testosterone derivative, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H] 19-nortestosterone for the estimation of cytosolic androgen receptors in male rat liver is described. Use of this compound demonstrates binding which has a similar dissociation constant, maximum binding and steroid specificity to that seen with other synthetic testosterone derivatives. In contrast to previous data significant binding to the progesterone receptor also occurs and future studies with this ligand should employ triamcinolone acetonide to block such binding.     

Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDH enzyme activity: physiological implications

In this study rat brain solubilized plasmalemma-microsomal fractions (B-P3) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BSA at C-3 position) and E-6-BSA (17beta-estradiol linked to BSA at C-6 position) affinity columns. It is interesting that a 37 kDa protein was retained by both columns which was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was not retained by either a control BSA conjugated affinity column or a corticosterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding affinity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17beta-E-6-BSA to GAPDH was impeded by free estrogen (17beta-estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle using radiolabeled ligand binding assays revealed that P-3-BSA had 10x lower GAPDH binding affinity than E-6-BSA. Next, the effects of estrogen and progesterone on GAPDH activity were studied. Rapid and significant increases in V(max) and changes in K(m) were observed by the addition of 10 nM estradiol, whereas 100 nM progesterone decreased only V(max) significantly. Testosterone, corticosterone, 17alpha-estradiol, and diethylstilbestrol did not affect the enzyme activity. The results indicate that GAPDH is a target site for 17beta-estradiol and progesterone and suggest possible roles in the regulation of cellular metabolism and synaptic remodeling in which GAPDH has been reported to be involved.     

The metabolism of progesterone by animal tissues in vitro. Sex and species differences in conjugate formation during the metabolism of [4-14C]progesterone by liver homogenates

1. Sex and species differences during the metabolism of [4-¹⁴C]progesterone by liver homogenates from rat, rabbit, guinea pig and hamster have been investigated. 2. Liver homogenate from male rat formed `water-soluble' metabolites faster and in significantly larger amounts than did liver homogenate from female rat. About 65–70% of the added progesterone was conjugated as glucuronide by liver homogenate from male rat and about 45–50% by that from female rat. Liver homogenate from male rat also formed glucuronides faster than did liver homogenate from female rat. Sulphate formation was low (8–16%) in liver homogenates from both male and female rats. 3. Hamster-liver homogenate did not show any sex difference in the rate of formation of `water-soluble' metabolites, but a sex difference was observed in the amount of free steroids recovered at low tissue:steroid ratios. Liver homogenate from female hamster formed glucuronides faster and in significantly larger amounts than did liver homogenate from male hamster, the reverse of what was found in rat liver. 4. Liver homogenates from male and female rabbits and guinea pigs formed `water-soluble' metabolites that were almost entirely glucuronides. 5. Neither rabbit liver nor guinea-pig liver showed any significant sex difference in the rate or amount of formation of total `water-soluble' metabolites or glucuronides, but guinea-pig liver was considerably less active than rabbit liver. 6. Glucuronides were quantitatively the major type of conjugate formed by the liver homogenates from both sexes of all species except the male hamster.     

Steroid hormones and the fertilizing capacity of spermatozoa

The effects of eleven different steroid hormones on $\text{in vitro}$ development of fertilizing capacity by hamster sperm were examined. Capacitation of epididymal sperm occurred only in the presence of female genital tract secretions. Fertilizing ability of sperm was poor if estradiol-17β, cortisol, chlormadinone acetate, medroxyprogesterone acetate, or megestrol acetate were present in the incubation medium at 10^?5M, whereas similar concentrations of estradiol-17α, progesterone, norethisterone acetate, ethynodiol diacetate, or norgestrel had little effect. Testosterone was a weak inhibitor of capacitation. Capacitation activity of female uterus and oviduct washings was higher at estrus than diestrus. This activity was reduced by treating intact animals with progesterone, cortisol, or testosterone, but increased by estradiol-17β or HCG. Estradiol-17α has no effect. Activity was low in pregnant or ovariectomized hamsters. Treatment of ovariectomized animals with estradiol-17β increased capacitation activity, but estradiol-17α, HCG or progesterone treatment was ineffective.     

Substrate-binding ability of Escherichia coli ribonucleic acid polymerase in relation to its protein composition.

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.     

Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme

Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.     

Effects of asbestos on membrane transport and metabolism of benzo(a)pyrene

The effect of asbestos on benzo(a)pyrene uptake by microsomal membranes and lipid micelles has been investigated. Asbestos mediates a rapid transport of the carcinogen into the membrane and also impairs benzo(a)pyrene metabolism in rabbit and rat liver microsomes by markedly inhibiting aryl hydrocarbon hydroxylase.     

Effects of clofibrate on the metabolism of progesterone and oestradiol in rat liver microsomal fraction

1. The substrate conversion of [4-(14)C]progesterone and [4-(14)C]oestradiol during incubation with the liver microsomal fraction from both control and clofibrate-treated rats amounted to about 10-15 and 20-25% respectively. 2. The metabolites of progesterone formed by preparations from control rats were hydroxylated in the 16alpha-position (14%), the 6beta-position (12%) and the 2alpha-position (7%). Of the products formed from oestradiol 12% were recovered as a 16alpha-hydroxylated derivative whereas 5% had a 6beta- and 2% a 6alpha-hydroxyl group. 3. Clofibrate affected the microsomal metabolism of both progesterone and oestradiol. It induced 7alpha-hydroxylation of both compounds, metabolic conversions not found in control rats. The 6beta-hydroxylation of progesterone and the 6alpha-hydroxylation of oestradiol were enhanced by a factor of 2 and 3.5 respectively. The 2alpha-hydroxylation, and the 20alpha- and 20beta-hydroxy steroid reduction of progesterone were significantly decreased as were the 16alpha- and the 6beta-hydroxylation of oestradiol.     

A dose-response study of forskolin, stimulatory hormone, and guanosine triphosphate analog on adenylate cyclase from several sources

We have described relationships involving forskolin stimulation of adenylate cyclase (AC) from a variety of sources and the potentiation of forskolin effects by stimulatory hormones (glucagon, ACTH, and epinephrine) and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p). The effects on AC were examined using membrane preparations of rabbit adipocytes, rat adipocytes, rat erythrocytes, and rat liver. Also examined was the AC of liver membranes of rat pretreated with pertussis toxin as well as that solubilized from rat liver membranes. Maximal forskolin stimulation of AC in all preparations studied revealed a consistent 10-fold increase in AC activity. The EC50 for forskolin was 10 microM for rat liver, 15 microM for rabbit and rat adipocytes and 17 microM for rat erythrocyte AC stimulation. In all cases the AC activity attained by forskolin stimulation was further enhanced by stimulatory hormones in a dose-dependent manner. Furthermore, a combination of all three activators (forskolin, stimulatory hormone, and Gpp(NH)p) resulted in an even greater overall stimulation to levels ranging from 25- to 30-fold over unstimulated activity levels. In the presence of saturating levels of each stimulatory hormone and Gpp(NH)p, the EC50 for forskolin diminished markedly to the range of 0.5 to 4.0 microM. In the absence of any apparent tissue specificity for forskolin stimulation, the general pattern of these results further implicates the catalytic site of the AC complex as the site of forskolin activation. Furthermore, activation of additional components of the complex by Gpp(NH)p and tissue specific hormones may further influence the AC activity and thereby potentiate the stimulation by forskolin.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.