Role of electron transport in the regulation of the lyase activity of C21 side-chain cleavage P-450 from porcine adrenal and testicular microsomes

The C21 side-chain cleavage enzymes from porcine adrenal and testicular microsomes have been purified and shown to resemble each other very closely (Nakajin, S., Shinoda, M., Hanui, M., Shively, J.E., and Hall, P.F. (1984) J. Biol. Chem. 259, 3971-3976). We have investigated the reason for the low levels of lyase activity shown by adrenal microsomes as compared to testicular microsomes. Competition for substrate with 21-hydroxylase in adrenal microsomes was excluded by studies showing that antibodies to 21-hydroxylase do not increase lyase activity in spite of almost complete inhibition of 21-hydroxylation. Reconstitution of the purified testicular enzyme in lipids extracted from adrenal and testicular microsomes excluded a specific effect of lipids on lyase activity. On the other hand, addition of porcine hepatic P-450 reductase to microsomes from adrenal and testis increased the activity of lyase relative to hydroxylase. The same effect is seen when reductase is added to the pure enzymes. As the concentration of reductase increases, lyase activity increases relative to hydroxylase until the rates of the activities become almost equal. Vmax is the same for both activities (hydroxylase and lyase) of the two enzymes (6.3-6.5 nmol/min/nmol of P-450). Km for reductase is approximately the same for the hydroxylase activities (0.4-0.6 microM) and for the lyase activities (1.7-2.0 microM) of the two enzymes. Antibodies to reductase, when added to testicular microsomes, inhibit both activities, but inhibition of lyase is greater than that of hydroxylase. The enzyme activity of reductase in testicular microsomes is 3-4 times higher than that of adrenal microsomes (0.29 and 0.08 nmol/min/mg of protein, respectively). These findings may account for the greater activity of lyase in testicular as opposed to adrenal microsomes.     

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.     

Microsomal cytochrome P-450 from neonatal pig testis: two enzymatic activities (17 alpha-hydroxylase and c17,20-lyase) associated with one protein

Studies have been performed to test the hypothesis that cytochrome P-450 from testicular microsomes consists of a single protein with two enzymatic activities (17 alpha-hydroxylase and C17,20-lyase). Three lines of evidence to support the hypothesis were obtained. (1) The enzyme appears to be homogeneous by immunochemical criteria with anti-P-450 IgG (line of identity on immunodiffusion and a single band on immunoelectrophoresis), by demonstration of a single NH2-terminal amino acid (methionine) and the finding of 16 single amino acids at the NH2 terminus. (2) Optima for pH and temperature are the same for both enzymatic activities (pH 7.25 and 37 degrees C), and temperatures between 30 and 44 degrees C decreased both activities in such a way that the ratio of hydroxylase to lyase was the same at all temperatures tested. (3) A variety of inhibitors affect both activities to the same extent: Ki values for two competitive inhibitors (SU 8000, 0.04 microM; SU 10603, 0.3 microM) are the same for hydroxylase and lyase; partition coefficients for inhibition by carbon monoxide are similar for hydroxylase and lyase (20 +/- 2 and 27 +/- 3); anti-P-450 (serum and IgG) causes inhibition of both activities to the same extent, and the same is true of a variety of less specific inhibitors. It is concluded that a single heme protein (cytochrome P-450) from microsomes of neonatal pig testis catalyzes two reactions (hydroxylase and lyase) which are sequential steps in the synthesis of androgens by the testis leading to conversion of C21 precursors to C19 steroid hormones.     

Contribution of cytochrome b5 to androgen synthesis in rat testicular microsomes

Cytochrome b5 was purified from porcine testicular microsomes, and its amino acid composition was determined. Rabbit antibody against the purified cytochrome b5 was prepared in order to study the contribution of cytochrome b5 to testicular microsomal oxygenases related to androgen production. In the presence of NADPH alone as the electron donor, the antibody against cytochrome b5 inhibited the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase of rat testicular microsomal fraction. Addition of NADH to the NADPH-supported oxygenase assay system enhanced both steroid oxygenase activities, and addition of the antibody against cytochrome b5 decreased the NADH-caused stimulation of steroid 17 alpha-hydroxylase and C-17-C-20 lyase activities. When dehydroepiandrosterone and NAD+ were added as substrates for 3 beta-hydroxy-delta 5-steroid dehydrogenase in order to synthesize NADH by enzymatic reaction, the NADPH-supported activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase were further stimulated as compared with the addition of NADH, and this stimulation was suppressed by the antibody against cytochrome b5. These results suggest that cytochrome b5, together with 3 beta-hydroxy-delta 5-steroid dehydrogenase, contributes to the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase in the testicular microsomal fraction.     

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.     

Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats.

When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.     

Destruction of testicular cytochrome P-450 by 7 alpha-thiospironolactone is catalyzed by the 17 alpha-hydroxylase.

Studies were done to determine the role of the 17 alpha-hydroxylase in the conversion of 7 alpha-thiospironolactone (7 alpha-thio-SL) to a reactive metabolite causing the degradation of testicular cytochrome P-450. Incubation of guinea pig testicular microsomes with 7 alpha-thio-SL plus NADPH resulted in an approx. 70% decline in cytochrome P-450 content and even greater loss of 17 alpha-hydroxylase activity. Addition of the 17 alpha-hydroxylase inhibitor, SU-10'603, to the incubation medium prevented the degradation of P-450 by 7 alpha-thio-SL. Similarly, preincubation of testicular microsomes with anti-P-45017 alpha,lyase IgG to inhibit 17 alpha-hydroxylation, diminished the subsequent loss of P-450 caused by 7 alpha-thio-SL. The results indicate that the 17 alpha-hydroxylase catalyzes the conversion of 7 alpha-thio-SL to the reactive metabolite responsible for P-450 destruction. The accompanying loss of 17 alpha-hydroxylase activity supports the hypothesis that suicide inhibition is the mechanism involved.     

Microsomal cytochrome P-450 from neonatal pig testis. Purification and properties of A C21 steroid side-chain cleavage system (17 alpha-hydroxylase-C17,20 lyase)

A cytochrome P-450 from neonatal pig testicular microsomes was purified to homogeneity as judged by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and by double diffusion on agar against antiserum raised in rabbits against the protein. The enzyme shows both 17 alpha-hydroxylase (Vmax = 4.6 nmol of product/min/nmol of P-450, Km = 1.5 microM) and C17,20 lyase (Vmax = 2.6 nmol of product/min/nmol of P-450, Km = 2.4 microM) activities. Both activities require NADPH and a flavoprotein P-450 reductase; microsomal P-450 reductase from pig and rat livers was used in these studies. The enzyme possesses a single subunit of molecular weight 59,000 +/- 1,000 as determined by electrophoresis on polyacrylamide with sodium dodecyl sulfate and by chromatography on sodium dodecyl sulfate-Sephadex. The enzyme is a glycoprotein and contains 8 nmol of heme/mg of protein and 40 nmol of phospholipid/mg of protein. All heme detected by pyridine hemochromogen is accounted for as P-450 by difference spectroscopy of the reduced P-450.carbon monoxide complex. This complex shows an absorbance maximum at 448 nm with no evidence of P-420. These studies raise the possibility that one microsomal protein (cytochrome P-450) may possess two enzymatic activities (hydroxylase and lyase).     

Cytochrome P-450 C21scc: One enzyme with two actions: Hydroxylase and lyase

Testis, adrenal, ovary and placenta contain a microsomal cytochrome P-450 that is capable of converting progesterone to androstenedione and pregnenolone to dehydroepi-androsterone. This conversion requires 17-hydroxylation followed by C_17,20-lyase activity which are both catalyzed by this one protein. Gene cloning and Northern blotting reveal that, at least in man, the same gene is responsible for both testicular and adrenal enzymes. The enzyme was first purified from neonatal pig testis. Both the testicular and adrenal enzymes show a marked preference for the 5-ene substrate (pregnenolone) in keeping with the extensive use of the 5-ene pathway in that species. Affinity alkylation with 17-bromoacetoxyprogesterone reveals a conserved cysteine at the active site of the enzyme and confirms the conclusion that a single enzyme catalyzes both reactions. Under some circumstances the enzyme catalyzes only 17-hydroxylation to permit the formation of the C₂₁ steroid cortisol. The regulation of lyase activity, i.e. the determination of the extent to which the second activity is expressed, results from the availability of P-450 reductase. No doubt the greater concentration of this protein in testicular as opposed to adrenal microsomes (× 3.5) is responsible for the production of androgens in the testis and cortisol in the adrenal. Testicular cytochrome b₅ also specifically stimulates lyase activity and also causes the porcine enzyme to catalyze a new reaction, i.e. Δ¹⁶-synthetase, resulting in synthesis of the important pheromone androsta-4,16-dien-3 one from progesterone.     

Purification and properties of steroid 17 alpha-hydroxylase from calf testis

Steroid 17 alpha-hydroxylase has emerged as a key enzyme in steroidogenic cells: (i) it represents the branch point between the 17-deoxy (mineralo) and the 17-hydroxy (gluco) corticosteroid pathways in the adrenal cortex; (ii) the corresponding specific cytochrome (P-450(17 alpha] is highly dependent upon hormonal regulation; and (iii) the enzyme also catalyzes the steroid 17-20 lyase reaction, leading to the major androgens in the testis. As a prerequisite to the study of its regulation in intact cell, 17 alpha-hydroxylase was purified from calf testis microsomal preparations. Following five chromatographic steps, the enzyme was obtained as an apparently homogeneous protein of Mr = 57 kDa upon gel electrophoresis. The procedure yielded a recovery of about 10% as judged by cytochrome P-450 assay. Whereas 17 alpha-hydroxylase specific activity was about 30-fold enriched during the purification, that of the C17-20 lyase was increased by about 6-fold, strongly suggesting that its organelle environment may modulate the enzymatic activity. The purified enzyme yielded a 20 N-terminal amino-acid sequence showing a complete homology with that of its adrenal counterpart and a polyclonal antibody raised against our preparation revealed a 57 kDa protein band in bovine adrenocortical microsomal extracts, upon immunoblotting experiments. It was thus concluded that bovine 17 alpha-hydroxylase activity is supported by highly similar if not identical enzymatic proteins in both testis and adrenal cortex tissues. The purified P-450(17 alpha) preparation is now being used in reconstitution experiments which suggest that microsomal components may contribute to a different expression of the enzyme specificity in its native testis or adrenocortical intracellular environment, respectively.     

Inhibitory effect of some imidazole antifungal compounds on the synthesis of 16-ene-C19-steroid catalyzed by pig testicular microsomes

The activity of the enzyme (16-ene-C19-steroid synthesizing enzyme) responsible for the conversion of C21-steroids to 16-ene-C19-steroids, which was localized on pig testicular microsomes, was inhibited by some typical imidazole antifungal compounds such as clotrimazole, econazole, miconazole and ketoconazole which are known to be universal inhibitors of cytochrome P-450-dependent enzymes. The 50% inhibitory concentrations of clotrimazole, econazole and miconazole were 0.29, 0.36 and 1.25 microM, respectively for 16-ene-C19-steroid synthesizing enzyme activity. Clotrimazole was the most powerful inhibitor of all the compounds examined, which shows the competitive inhibition for 16-ene-C19-steroid synthesizing enzyme activity. The Ki-value was 0.26 microM for its activity. The degree of the inhibition by these imidazole compounds was very similar to the inhibition of 17 alpha-hydroxylase and C17,20-lyase activities on pig testicular microsomes.     

The interference of polychlorinated biphenyls (Aroclor 1254) with membrane regulation of the activities of cytochromes P-450C21 and P-450(17) alpha,lyase in guinea-pig adrenal microsomes.

Regulation of cytochromes P-450 21-hydroxylase (P-450C21) and P-450 17 alpha-hydroxylase/C17,20-lyase (P-450(17) alpha,lyase) activities and impairment of this regulation by Aroclor 1254 was studied in guinea-pig adrenal microsomes. In a membrane depleted system, a decrease in the normally predominant, P-450C21 activity and an increase in P-450(17) alpha,lyase activities was observed. The same deviations were observed in intact microsomes with increase in the reaction temperature (0-40 degrees C). Breaks in Arrhenius plots for activities of P-450C21 and P-450(17) alpha,lyase correlate with transition temperatures reported for the microsomal membrane. These results point to: (1) preference of a gel state membrane for catalytic expression of P-450C21 suggesting a clustered organization of this P-450 species with reductase; (2) preference of a fluid membrane for lyase activity suggesting a random collision mechanism for reduction of P-450(17) alpha,lyase. Aroclor 1254 introduced to reaction mixtures containing intact microsomes elicited basically the same changes as caused by depletion of the microsomal membrane or by increase in the incubation temperature. Lack of effect of Aroclor 1254 on P-450C21 and P-450(17) alpha,lyase activities in the membrane depleted system demonstrates that its interference with monooxygenase activities is mediated by the microsomal membrane. The similarities between altered cytochrome P-450 mediated activities in the presence of Aroclor 1254 and the deviations observed in the membrane depleted system or upon increase in the incubation temperature may suggest that this chemical exerts its impacts by influencing membrane fluidity.     

Ovine steroid 17alpha-hydroxylase cytochrome P450: characteristics of the hydroxylase and lyase activities of the adrenal cortex enzyme

The steroid 17-hydroxylase cytochrome P450 (CYP17) found in mammalian adrenal and gonadal tissues typically exhibits not only steroid 17-hydroxylase activity but also C-17,20-lyase activity. These two reactions, catalyzed by CYP17, allow for the biosynthesis of the glucocorticoids in the adrenal cortex, as a result of the 17-hydroxylase activity, and for the biosynthesis of androgenic C(19) steroids in the adrenal cortex and gonads as a result of the additional lyase activity. A major difference between species with regard to adrenal steroidogenesis resides in the lyase activity of CYP17 toward the hydroxylated intermediates and in the fact that the secretion of C(19) steroids takes place, in some species, exclusively in the gonads. Ovine CYP17 expressed in HEK 293 cells converts progesterone to 17-hydroxyprogesterone and pregnenolone to dehydroepiandrosterone via 17-hydroxypregnenolone. In ovine adrenal microsomes, minimal if any lyase activity was observed toward either progesterone or pregnenolone. Others have demonstrated the involvement of cytochrome b(5) in the augmentation of CYP17 lyase activity. Although the presence of cytochrome b(5) in ovine adrenocortical microsomes was established, ovine adrenal microsomes did not convert pregnenolone or 17-hydroxypregnenolone to dehydroepiandrosterone. Furthermore the addition of purified ovine cytochrome b(5) to ovine adrenal microsomes did not promote lyase activity. We conclude that, in the ovine adrenal cortex, factors other than cytochrome b(5) influence the lyase activity of ovine CYP17.     

Stimulatory effect of soluble supernatant on hydroxylase activity of rat testis microsomes.

Addition of soluble supernatant to testis microsomes results in 42% increase in steroid 17,20-lyase activity and a 65% increase in 17alpha-hydroxylase activity. This stimulatory activity could be partially purified by salt fractionation. The activating factor(s) was not removed by dialysis nor did it appear to be lipid. It was destroyed by trypsin. Differential effects of heat were observed with the hydroxylase and lyase activators. The activation did not affect Km but only increased Vmax. The supernatant could be added to each enzyme to the point of maturation. No binding of steroids by the supernatant could be detected. Corpus luteum and placental supernatant did not stimulate enzymic activity, but supernatant from an adrenal adenoma was active.     

Production of testosterone from progesterone by rat testicular microsomes without release of the intermediates 17 alpha-hydroxyprogesterone and androstenedione

It has been shown that during the in vitro conversion of progesterone to androstenedione, 17 alpha-hydroxyprogesterone is not an obligatory intermediate which equilibrates with freely diffusible steroids in the incubation medium. Recently a cytochrome P-450 was purified that catalyzed, in addition to hydroxylase/lyase activities, reduction of androstenedione to testosterone. In order to determine whether progesterone could be transformed to testosterone without both intermediates (17 alpha-hydroxyprogesterone and androstenedione) being equilibrated with steroids in the medium, several double-label double-substrate experiments were performed. When rat microsomes were incubated with an equimolar mixture of [14C]progesterone and 17 alpha-hydroxy[3H]progesterone, androstenedione was isolated with a 11-fold higher 14C/3H ratio than 17 alpha-hydroxyprogesterone, indicating that androstenedione could not be produced from free, diffusible 17 alpha-hydroxyprogesterone. Incubation of an equimolar mixture of 17 alpha-hydroxy[3H]progesterone and [14C]androstenedione with testicular microsomes resulted in the incorporation of 3-4-fold more 17 alpha-hydroxyprogesterone into testosterone than of androstenedione, although the latter is the immediate precursor of testosterone. In an experiment in which equimolar concentrations of [3H]progesterone and [14C]androstenedione were incubated with testicular microsomes, the large pool of progesterone inhibited competitively lyase activity, but still the label of progesterone was incorporated into testosterone to the same extent as that of androstenedione. These results indicate that testosterone can be produced by immature rat testicular microsomes from added progesterone on an organized unit without the intermediates equilibrating with the incubation medium.     

Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex.

We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.     

Xenopus laevis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5

The enzyme CYP17 primarily regulates androgen production by mediating four reactions: conversion of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively (17alpha-hydroxylase activity), followed by conversion of the 17-hydroxylated steroids to dehydroepiandrosterone and androstenedione, respectively (17,20-lyase activity). Most mammalian CYP17 isoforms have high 17alpha-hydroxylase relative to 17,20-lyase activities and preferentially mediate one of the two 17,20-lyase reactions. In contrast, Xenopus laevis CYP17 potently regulates all four reactions in the frog ovary. CYP17 isoforms generally rely on the cofactor cytochrome b(5) for the 17,20-lyase reaction, suggesting that the high lyase activity of Xenopus CYP17 might be due to a lesser dependence on b(5). The kinetics of Xenopus CYP17 expressed in yeast microsomes were therefore examined in the absence and presence of Xenopus on human b(5). Xenopus CYP17 mediated both 17,20-lyase reactions in the absence of b(5), confirming that the activity did not require b(5). However, both Xenopus and human b(5) slightly enhanced Xenopus CYP17-mediated lyase activity, indicating that the enzyme was still at least partially responsive to b(5). Surprisingly, only the human b(5) cofactor enhanced human CYP17-mediated lyase activity, implying that the human enzyme had more specific cofactor requirements than Xenopus CYP17. Studies using human/Xenopus chimeric b(5) proteins revealed that human b(5) residues 16-41 were important for the specific regulation of the lyase activity of HuCYP17, possibly serving as an interacting domain with the enzyme. CYP17 may therefore have evolved from a general producer of sex steroids in lower vertebrates to a more tightly regulated producer of both sex steroids and glucocorticoids in mammals.     

Kinetic control of steroidogenesis by steroid concentration in guinea pig adrenal microsomes

For clarification of the effects of steroid concentration on steroidogenesis of adrenal microsomes, the kinetic parameters, Km and kcat, were determined in the steady-state for progesterone and 17 alpha-hydroxyprogesterone metabolism catalyzed by P-450C21 and P-450(17 alpha lyase) in guinea pig adrenal microsomes. At a high concentration of progesterone, it was equally metabolized by P-450C21 and P-450(17 alpha lyase), while at a low concentration, it was hydroxylated at 17 alpha-position with twice higher rate than at 21-position. 17 alpha-Hydroxyprogesterone is apparently metabolized preferentially by P-450C21 at any concentration. Although the productions of deoxycortisol and androstenedione from 17 alpha-hydroxyprogesterone were strongly inhibited by progesterone, androstenedione formation from progesterone was not inhibited by a high concentration of progesterone. The addition of liposomal P-450C21 to the reaction medium containing adrenal microsomes caused a decrease in the concentration of 17 alpha-hydroxyprogesterone released into the medium in the steady state reaction, but this had no effect on the activity of androstenedione formation from high concentrations of progesterone. It thus follows that androstenedione is produced by successive monooxygenase reactions without the release of 17 alpha-hydroxyprogesterone from P-450(17 alpha lyase) at a high concentration of progesterone, which is the condition of the adrenal microsomes in vivo.