Cytochrome P-450(17 alpha,lyase)-mediating pathway of androgen synthesis in bovine adrenocortical cultured cells.

Cytochrome P-450(17 alpha,lyase) mediating pathway of dehydroepiandrosterone (DHA) formation from pregnenolone was investigated in primary cultures of bovine adrenocortical fasciculata-reticularis cells. To determine whether DHA formation proceeds predominantly by successive monooxygenase reactions without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) the cells were incubated with [14C]pregnenolone and 17 alpha-[3H]hydroxypregnenolone in the presence of Trilostane. Results of the double-substrate double-label experiments indicate that in the presence of high concentration of pregnenolone most of DHA was formed, directly from pregnenolone by the successive reactions. Since the concentration of pregnenolone usually exceeds that of 17 alpha-hydroxypregnenolone in the adrenal glands, DHA is concluded to be formed predominantly by successive reactions from pregnenolone without 17 alpha-hydroxypregnenolone leaving P-450(17 alpha,lyase) in vivo. By chronic ACTH treatment, the activities of 17 alpha-hydroxylation and DHA formation in adrenocortical cultured cells became higher concomitantly with the increase of P-450(17 alpha,lyase) content. Most of DHA was found to be formed by successive reactions from pregnenolone even under such conditions.     

Induction of 17 alpha-hydroxylase (cytochrome P-450(17)alpha) activity by adrenocorticotropin in bovine adrenocortical cells maintained in monolayer culture

Using bovine adrenocortical cells in monolayer culture it has been shown that treatment with adrenocorticotropin (ACTH) causes a dramatic increase in 17 alpha-hydroxylase activity. In postmitochondrial supernatant fractions (PMS) prepared from cells maintained in culture, there was a 15-fold increase in 17 alpha-hydroxylase activity 36 h following initiation of ACTH treatment compared with the activity measured in PMS prepared from control cells. In the continued presence of ACTH, 17 alpha-hydroxylase activity declined; however, even after 60 h of exposure to ACTH, 17 alpha-hydroxylase activity was eight times higher than that present in control cells. The dramatic increase in 17 alpha-hydroxylase activity provides an explanation for the previously observed phenomenon that following initiation of ACTH treatment of bovine adrenocortical cells in monolayer culture there is a shift in the pattern of corticosteroid secretion from approximately equal amounts of cortisol and corticosterone to almost exclusively cortisol. Thus, the modulation of 17 alpha-hydroxylase activity by ACTH action appears to serve a key regulatory role in the pattern of corticosteroid production. Soluble cytosolic factors apparently do not participate in the regulation of 17 alpha-hydroxylase activity in the bovine adrenal cortex. Increases in the magnitude of substrate-induced absorbance changes are indicative that the increase in 17 alpha-hydroxylase activity is due, at least in part, to an elevation of cytochrome P-450(17)alpha synthesis.     

Purification of cytochromes P-450(scc) and P-450(17 alpha) by steroid-binding affinity column chromatography.

The preparation, testing and use of a variety of cholesterol-, deoxycorticosterone (DOC)- and pregnenolone-binding 1,6-diaminohexyl (EAH)-Sepharose 4B supports for affinity column chromatography of cytochromes P-450(scc) and P-450(17 alpha) from bovine adrenal and pig testis are described. EAH-Sepharose 4B has free amino groups at the end of a 10-atom spacer arm. Hydroxyl groups of cholesterol (3 beta), deoxycorticosterone (21 beta) and pregnenolone (3 beta) are linked to succinic anhydride in pyridine through an ester linkage. These coupling ligands of hemisuccinate were synthesized by a general procedure. Free amino groups of EAH-Sepharose 4B were used to couple ligands, containing carboxyl groups, by the carbodiimide coupling method. Both the purified cytochromes P-450(scc) and P-450(17 alpha) were found to be homogeneous and estimated to have a molecular weight of 52,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The carbon monoxide difference spectra with peaks at 450 and 448 nm exhibit the absorption spectra of typical cytochromes P-450(scc) and P-450(17 alpha), respectively. Cytochromes P-450(scc) and P-450(17 alpha) were determined to have isoelectric points of 8.0 and 6.5 in isoelectric focusing on a pH gradient gel. Cytochrome P-450s can be purified between 425- and 1000-fold from the crude extracts.     

Effect of spin state on reduction of cytochrome P-450 (P-450C21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450C21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17 alpha-hydroxyprogesterone-bound P-450C21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25 degrees C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450C21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25 degrees C. The steady-state results showed that the rate of P-450C21 reduction was not affected by the high spin content of substrate-bound P-450C21 but was very slow without a steroid substrate. Biphasic reduction of P450C21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17 alpha-hydroxyprogesterone-bound and progesterone-bound P-450C21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450C21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450C21.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.     

The active form of cytochrome P-450 from bovine adrenocortical mitochondria.

Cytochrome P-450 from bovine adrenocortical mitochondria exists in three forms of molecular weight: 850,000 (protein 16), of one-half (protein 8), and of one-quarter of this value (protein 4). The forms of the enzyme are named according to the number of subunits and all appear to be active in converting cholesterol to 3beta-hydroxy-5-pregnen-20-one (side chain cleavage) (Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5606). To determine whether all three forms are active at their characteristic molecular weights, the three cytochromes were each layered onto separate sucrose density gradients and centrifuged at 49,000 rpm for 60 min; the gradients contained all the factors necessary for side chain cleavage including one of the following substrates: cholesterol, 20S-hydroxycholesterol, and 20S,22R-dihydroxycholesterol. Regardless of the form of P-450 layered onto the gradient and regardless of the substrate, enzyme activity (side chain cleavage) was observed only in fractions corresponding to a sedimentation coefficient of 20 to 22 S which is that for protein 16. No activity was observed at S values corresponding to either protein 8 or protein 4. These findings indicate that the active form of cytochrome P-450 from adrenocortical mitochondria is that containing 16 subunits, i.e. the form in which the cytochrome is normally isolated from adrenal mitochondria. Forms consisting of eight and four subunits which can be prepared from protein 16 become active only by forming protein 16, at least in an aqueous medium in vitro.     

Synthesis and processing of mitochondrial steroid hydroxylases. In vivo maturation of the precursor forms of cytochrome P-450scc, cytochrome P-450(11)beta, and adrenodoxin

The synthesis and maturation of the precursor forms of three mitochondrial enzymes involved in steroid hormone biosynthesis have been studied in vivo. Primary cultures of bovine adrenocortical cells were radiolabeled with [35S] methionine and newly synthesized cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta), and adrenodoxin immunoisolated using specific antibodies. Both the precursor and mature forms of P-450scc and P-450(11)beta were detected during short periods of pulse labeling; however, the precursor forms were transitory in nature while their corresponding mature forms accumulated. Pulse-chase experiments showed that the precursor form of each cytochrome P-450 had an apparent half-life of 3.5 min. In contrast, the precursor form of adrenodoxin was not readily detected in pulse-labeling experiments until a substantial amount of its mature form had accumulated. When the cultured cells were treated with a chelator of divalent cations (o-phenanthroline) or a mitochondrial uncoupler (dinitrophenol), the maturation of all three precursors was inhibited. The synthesis of the P-450scc and P-450(11)beta precursors was induced in cells maintained in the presence of adrenocorticotropin, and the rates of appearance of their processed forms were also increased. The mature forms of all three proteins were immunoisolated from a trypsinized mitochondrial fraction prepared from the radiolabeled cells, demonstrating that the mature proteins were localized within the organelle. These studies establish that the maturation of the precursor forms of the mitochondrial steroidogenic enzymes are characterized by steps similar to those reported for other mitochondrial precursor proteins.     

Organ-specific properties of cytochromes P-450s21 (steroid 21-hydroxylases) of liver and adrenocortical microsomes

Prothrombin, plasminogen, urokinase- and tissue-type plasminogen activators contain homologous structures known as kringles . The kringles correspond to autonomous structural and folding domains which mediate the binding of these multidomain proteins to other proteins. During evolution the different kringles retained the same gross architecture, the kringle -fold, yet diverged to bind different proteins. We show that the amino acid sequences of the type II structures of the gelatin-binding region of fibronectin are homologous with those of the protease- kringles . Prediction of secondary structures revealed a remarkable agreement in the positions of predicted beta-sheets, suggesting that the folding of kringles and type II structures may also be similar. As a corollary of this finding, the disulphide-bridge pattern of type II structures is shown to be homologous to that in kringles . It is noteworthy that protease- kringles and fibronectin type II structures have similar functions inasmuch as they mediate the binding of multidomain proteins to other proteins. It is proposed that the kringles of proteases and type II structures of fibronectin evolved from a common ancestral protein binding module.     

Participation of cytochrome P-450 in the steroid 17alpha-hydroxylase of testis microsomes.

The effect of metyrapone on the activity of the steroid 17alpha-hydroxylase from rat testis was evaluated. A competitive pattern of inhibition was observed after analysis of data using a least mean squares computer analysis. The substrate for the hydroxylase induced a Type I difference spectrum in an active suspension of Triton treated microsomes. The magnitude of this spectral change was dependent on steroid concentration and was diminished by metyrapone. The effect of metyrapone was abolished at infinite steroid concentration. These results confirm the participation of cytochrome P-450 as a reactant in the 17alpha-hydroxylase reaction.     

Cytochrome P-450(17alpha) in beta-cells of rat pancreas and its local steroidogenesis

We have found cytochrome P-450(17alpha) in the islets of Langerhans of rat pancreas. Its existence coincided with that of insulin and demarcated those of glucagon and somatostatin, demonstrating the localization in beta-cells. The enzyme has not only 17alpha-hydroxylase activity but also lyase one, which is a prerequisite for androgen biosynthesis. The pancreatic microsomes converted progesterone mainly to androstenedione with a minor production of 17alpha-hydroxyprogesterone. Due to a low activity of the built-in lyase, cytochrome P-450(17alpha) requires a sufficient electron-transfer from P-450 reductase or presence of an activator to promote the C-C bond cleavage. In beta-cells, P-450 reductase was abundant and could efficiently transfer electrons to P-450(17alpha). Actually, inhibition with anti-P-450 reductase or limitation of NADPH preferentially reduced the lyase activity. Androstenedione was accumulated when its further metabolism was suppressed. We also found localization of cytochrome P-450scc and 3beta-hydroxysteroid dehydrogenase in beta-cells. These results indicate that the immediate substrate for androgen formation, progesterone, is intracellularly produced and is converted mainly to androstenedione with support by an efficient electron supply from P-450 reductase. The product was supposed to be further metabolized to the reduced derivatives such as testosterone, 5alpha-androstanedione, and dihydrotestosterone, which would act as local steroids in the islets of Langerhans.     

Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Inhibition of bovine adrenocortical mitochondrial cytochrome P-450(11)beta-mediated reactions by imidazole derivatives and mineralocorticoid analogs

The effects of several imidazole antimycotic agents, an imidazole and several mineralocorticoid analogs on the cytochrome P-450(11)beta-catalyzed 11 beta-hydroxylation of 11-deoxycorticosterone and aldosterone synthesis were examined. Ketoconazole, clotrimazole, miconazole and etomidate were found to be potent inhibitors of the reactions, causing 50% inhibition of the 11 beta-hydroxylase activity at concentrations between 10(-8) and 10(-7) M. The potency of etomidate as to the inhibition of aldosterone- and 18-hydroxycorticosterone-production was found to be almost equal to that in the case of 11 beta-hydroxylation. Spironolactone and other newly synthesized mineralocorticoid analogs were also found to inhibit the cytochrome P-450(11)beta-mediated reactions. The ID50 values of these drugs for inhibition of the 11 beta-hydroxylase activity were almost equal to those in the case of the aldosterone- and 18-hydroxycorticosterone-biosynthetic activities. The results of kinetical studies indicated that one of the mineralocorticoid analogs, Compound 23-0586, acts as a competitive inhibitor for the cytochrome P-450(11)beta-mediated reactions.