Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta.

The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate-determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two-fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome.     

Placental cytochrome P450scc (CYP11A1): comparison of catalytic properties between conditions of limiting and saturating adrenodoxin reductase

The mitochondrial side-chain cleavage of cholesterol, catalysed by cytochrome P450scc, is rate-limiting in the synthesis of progesterone by the human placenta. Cytochrome P450scc activity is in turn limited by the concentration of adrenodoxin reductase (AR) in placental mitochondria. In order to better understand which components of the cholesterol side-chain cleavage system are important in the regulation of placental progesterone synthesis, we have examined their effects on P450scc activity with both saturating and limiting concentrations of AR. The present study reveals that decreasing the AR concentration causes a decrease in the K(m) of cytochrome P450scc for cholesterol, facilitating saturation of the enzyme with its substrate. Decreasing AR resulted in P450scc activity becoming less sensitive to changes in P450scc concentration. The adrenodoxin (Adx) concentration in mitochondria from term placentae is near-saturating for P450scc and under these conditions, we found that decreasing AR reduces the K(m) of P450scc for adrenodoxin. Increasing either the cholesterol or P450scc concentration increased the amount of AR required for P450scc to work at half its maximum velocity. A relatively small increase in AR can support considerably higher rates of side-chain cleavage activity when there is a coordinate increase in AR and P450scc concentrations. We conclude from this study that cholesterol is near-saturating for cytochrome P450scc activity in placental mitochondria due to the P450scc displaying a low K(m) for cholesterol resulting from the low and rate-limiting concentration of AR present. This study reveals that it is unlikely that cholesterol or adrenodoxin concentrations are important regulators of placental progesterone synthesis but AR or coordinate changes in AR and P450scc concentrations are likely to be important in its regulation.     

Cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria and human placenta: immunochemical properties and structural characteristics

An immunochemical comparison of components of cholesterol side chain cleavage system from bovine adrenocortical and human placental mitochondria has been carried out. Antibodies against cytochrome P-450scc, adrenodoxin reductase and adrenodoxin from bovine adrenocortical mitochondria were shown to cross-react with corresponding antigens of human placental mitochondria. A highly sensitive immunochemical method for cytochrome P-450scc determination has been developed. Limited proteolysis of cytochrome P-450scc of human placental mitochondria was studied, and the products of trypsinolysis were identified using antibodies against cytochrome P-450scc and fragments of its polypeptide chain: F1, F2 and F3. Immunochemical relatedness of ferredoxins from bovine adrenocortical and human placental mitochondria allowed one to develop a fast and efficient method for cytochrome P-450scc purification from human placental mitochondria by affinity chromatography on adrenodoxin-Sepharose.     

A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin.

Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.     

Purification and immunological characterization of human placental mitochondrial cytochrome P-450

Human placental mitochondrial cytochrome P-450 was purified to electrophoretic homogeneity by hydrophobic, anion exchange and cation exchange column chromatography. The specific content of the purified protein was 15.7 nmol/mg protein and it showed a single band mol. wt 48,000 D in SDS-gel electrophoresis. When reconstituted with bovine adrenal adrenodoxin reductase and adrenodoxin it converted cholesterol to pregnenolone (cholesterol side-chain cleavage activity, CSCC) at the rate of 1 pmol/min/pmol P-450. Antibodies against the purified protein were raised in rabbits. Inhibition studies demonstrated 85% inhibition of placental CSCC activity at an antibody/protein ratio of 10:1. Placental microsomal aromatase activity was inhibited by 47% at the same antibody/protein ratio. The antibody inhibited bovine mitochondrial CSCC activity by 87% at the same antibody/protein ratio. Placental microsomal 7-ethoxycoumarin O-deethylase, aryl hydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities were not significantly inhibited by the antibody. The results indicate that the purified protein catalyzes cholesterol side-chain cleavage reaction, human placental microsomal aromatase and bovine adrenal mitochondrial P-450scc may share common antigenic determinants with placental P-450scc, but the placental microsomal xenobiotic-metabolizing cytochrome(s) is (are) distinctly different.     

3-methoxybenzidine: A potent inhibitor of cholesterol side chain cleavage cytochrome P-450

The effect of 3-methoxybenzidine on the conversion of cholesterol to pregnenolone was investigated using a reconstituted enzyme system comprised of adrenodoxin, adrenodoxin reductase and cytochrome P-450scc purified from bovine adrenal cortex. Under conditions where the cytochrome P-450scc concentration was rate-limiting, 3-methoxybenzidine was found to be a potent inhibitor, causing 50% inhibition at 7 μM when using a cholesterol concentration of 70 μM. The parent compound, benzidine, was much less effective, exhibiting an Icn value of approximately 40 μM. No effect of 3-methoxybenzidine was observed on the adrenodoxin reductase and adrenodoxin-catalyzed reduction of cytochrome c by NADPH, and it is concluded that 3-methoxybenzidine acts on cytochrome P-450scc in inhibiting cholesterol side chain cleavage.     

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.     

Adrenodoxin with a COOH-terminal deletion (des 116-128) exhibits enhanced activity

Adrenodoxin, purified from bovine adrenal cortex, was subjected to trypsin cleavage to yield a trypsin-resistant form, designated TT-adrenodoxin. Sequencing with carboxypeptidase Y identified the trypsin cleavage site as Arg-115, while Edman degradation indicated no NH2-terminal cleavage. Native adrenodoxin and TT-adrenodoxin exhibited similar affinity for adrenodoxin reductase as determined in cytochrome c reductase assays. In side chain cleavage assays using cytochrome P-450scc, however, TT-adrenodoxin demonstrated greater activity than adrenodoxin with cholesterol, (22R)-22-hydroxycholesterol, or (20R,22R)-20,22-dihydroxycholesterol as substrate. This enhanced activity is due to increased affinity of TT-adrenodoxin for cytochrome P-450scc; TT-adrenodoxin exhibits a 3.8-fold lower apparent Km for the conversion of cholesterol to pregnenolone. TT-Adrenodoxin was also more effective in coupling with cytochrome P-450(11) beta, exhibiting a 3.5-fold lower apparent Km for the 11 beta-hydroxylation of deoxycorticosterone. In the presence of partially saturating cholesterol, TT-adrenodoxin elicited a type I spectral shift with cytochrome P-450scc similar to that induced by adrenodoxin, and spectral titrations showed that oxidized TT-adrenodoxin exhibited a 1.5-fold higher affinity for cytochrome P-450scc. These results establish that COOH-terminal residues 116-128 are not essential for the electron transfer activity of bovine adrenodoxin, and the differential effects of truncation at Arg-115 on interactions with adrenodoxin reductase and cytochromes P-450 suggest that the residues involved in the interactions are not identical.     

细胞色素P450scc在人早期胎盘中的表达（简报）

人妊娠期间,胎盘合成大量的类固醇激素,与妊娠的启动、维持、分娩以及胎儿的发育均存在密切的关系。阐明胎盘类固醇激素特别是孕酮合成与分泌的调节机制对于寻找理想的生育调控技术和生殖保健方法具有重要的意义。因此,胎盘类固醇激素合成与分泌的调节向来是生殖生物学与妇产科学领域所关注的焦点问题之一,     

Covalently crosslinked complexes of bovine adrenodoxin with adrenodoxin reductase and cytochrome P450scc. Mass spectrometry and Edman degradation of complexes of the steroidogenic hydroxylase system.

NADPH-dependent adrenodoxin reductase, adrenodoxin and several diverse cytochromes P450 constitute the mitochondrial steroid hydroxylase system of vertebrates. During the reaction cycle, adrenodoxin transfers electrons from the FAD of adrenodoxin reductase to the heme iron of the catalytically active cytochrome P450 (P450scc). A shuttle model for adrenodoxin or an organized cluster model of all three components has been discussed to explain electron transfer from adrenodoxin reductase to P450. Here, we characterize new covalent, zero-length crosslinks mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide between bovine adrenodoxin and adrenodoxin reductase, and between adrenodoxin and P450scc, respectively, which allow to discriminate between the electron transfer models. Using Edman degradation, mass spectrometry and X-ray crystallography a crosslink between adrenodoxin reductase Lys27 and adrenodoxin Asp39 was detected, establishing a secondary polar interaction site between both molecules. No crosslink exists in the primary polar interaction site around the acidic residues Asp76 to Asp79 of adrenodoxin. However, in a covalent complex of adrenodoxin and P450scc, adrenodoxin Asp79 is involved in a crosslink to Lys403 of P450scc. No steroidogenic hydroxylase activity could be detected in an adrenodoxin -P450scc complex/adrenodoxin reductase test system. Because the acidic residues Asp76 and Asp79 belong to the binding site of adrenodoxin to adrenodoxin reductase, as well as to the P450scc, the covalent bond within the adrenodoxin-P450scc complex prevents electron transfer by a putative shuttle mechanism. Thus, chemical crosslinking provides evidence favoring the shuttle model over the cluster model for the steroid hydroxylase system.     

Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain

A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.     

Interaction of Catalytic Domains in Cytochrome P450scc–Adrenodoxin Reductase–Adrenodoxin Fusion Protein Imported into Yeast Mitochondria

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc–adrenodoxin reductase–adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.     

Actions of cyclic adenosine monophosphate on the cytodifferentiation of ovarian cells: studies in cultured swine granulosa cells using a novel exogenous adenylate cyclase from Bordetella pertussis

The regulation by cAMP of cholesterol side-chain cleavage activity and the synthesis of immunoisolated cytochrome P-450scc and adrenodoxin proteins was investigated in primary cultures of swine ovarian (granulosa) cells. Administration of a novel adenylate cyclase toxin isolated from Bordetella pertussis increased granulosa-cell cAMP accumulation up to 200-fold over basal. These effects were additive with those of FSH, forskolin, and cholera toxin. In contrast, bacterial extracts BP 347 and BP 348 from mutant strains of B. pertussis that lack either all virulent factors or the adenylate cyclase toxin and hemolysin were devoid of effect. Granulosa-cell cAMP accumulation supported by active bacterial adenylate cyclase was accompanied by 2- to 11-fold, time-dependent increases in [35S]methionine incorporation into immunospecific cytochrome P-450scc and adrenodoxin. These increases in the synthesis of cholesterol side-chain cleavage proteins were associated with enhanced pregnenolone production in response to exogenous sterol substrate, 25-hydroxycholesterol, and augmented progesterone secretion both in the absence and presence of exogenous lipoprotein. Moreover, the effects of Bordetella adenylate cyclase toxin on granulosa cell steroidogenesis were functionally integrated with other regulatory responses, since the non-cAMP dependent effector, estradiol 17-beta, interacted synergistically with bacterial adenylate cyclase in stimulating progesterone production. We conclude that exogenous adenylate cyclase isolated from B. pertussis can be functionally integrated into the cAMP-dependent effector pathway of granulosa cells with a resulting increase in intracellular cAMP concentrations, augmented biosynthesis of progesterone and pregnenolone, enhanced synthesis of immunospecific cytochrome P-450scc and adrenodoxin, and synergistic interactions with a non-cAMP-dependent ovarian effector hormone (estradiol).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative structural-functional characterization of recombinant and natural adrenodoxin. Interaction with cytochrome P450scc.

The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.     

The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. F?rster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.     

Regulation of steroidogenesis in rat Leydig cells in culture: effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt2cAMP (1 mM) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt2cAMP was also observed. Both hCG and Bt2cAMP increased the rates of synthesis of cytochrome P-450scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt2cAMP-treated cells the rate of synthesis of cytochrome P-450scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450scc and its associated protein, adrenodoxin. Since Bt2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450scc and adrenodoxin are mediated by increased cyclic AMP formation.     

Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal 5'-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc

Covalent modification of cytochrome P-450scc (purified from bovine adrenocortical mitochondria) with pyridoxal 5'-phosphate (PLP) was found to cause inhibition of the electron-accepting ability of this enzyme from its physiological electron donor, adrenodoxin, without conversion to the "P-420" form. Reaction conditions leading to the modification level of 0.82 and 2.85 PLP-Lys residues per cytochrome P-450scc molecule resulted in 60% and 98% inhibition, respectively, of electron-transfer rate from adrenodoxin to cytochrome P-450scc (with beta-NADPH as an electron donor via NADPH-adrenodoxin reductase and with phenyl isocyanide as the exogenous heme ligand of the cytochrome). It was found that covalent PLP modification caused a drastic decrease of cholesterol side-chain cleavage activity when the cholesterol side-chain cleavage enzyme system was reconstituted with native (or PLP-modified) cytochrome P-450scc, adrenodoxin, and NADPH-adrenodoxin reductase. Approximately 60% of the original enzymatic activity of cytochrome P-450scc was protected against inactivation by covalent PLP modification when 20% mole excess adrenodoxin was included during incubation with PLP. Binding affinity of substrate (cholesterol) to cytochrome P-450scc was found to be increased slightly upon covalent modification with PLP by analyzing a substrate-induced spectral change. The interaction of adrenodoxin with cytochrome P-450scc in the absence of substrate (cholesterol) was analyzed by difference absorption spectroscopy with a four-cuvette assembly, and the apparent dissociation constant (Ks) for adrenodoxin binding was found to be increased from 0.38 microM (native) to 33 microM (covalently PLP modified).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the presence of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin in fresh granulosa cells. Effects of follicle-stimulating hormone and cyclic AMP on cholesterol side chain cleavage cytochrome P-450 synthesis and activity

The synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and adrenodoxin was studied both in freshly harvested bovine granulosa cells and in granulosa cells maintained in primary monolayer culture. In addition, the action of follicle-stimulating hormone (FSH) and cyclic AMP analogs to stimulate the synthesis of cytochrome P-450scc was investigated in cultured cells. Precursor forms of cytochrome P-450scc and adrenodoxin were immunoisolated from a cell-free translation system directed by RNA prepared from freshly obtained granulosa cells that were not luteinized. Furthermore, the presence of cytochrome P-450scc in lysates of granulosa cells freshly obtained from very small follicles (containing less than 0.1 ml of follicular fluid) and in mitochondria of freshly obtained granulosa cells was demonstrated by using an immunoblotting technique. Continuous treatment of cultured granulosa cells with FSH or with cyclic AMP analogs (dibutyryl cyclic AMP or 8-bromo cyclic AMP) for 72 h increased incorporation of [35S]methionine into immunoprecipitable cytochrome P-450scc. Moreover, FSH, dibutyryl cyclic AMP, and 8-bromo cyclic AMP stimulated pregnenolone production by cultured granulosa cells (2.3-, 4.0-, and 7.5-fold increase over control, respectively), indicative of an increase in cholesterol side chain cleavage activity. The results of this study demonstrate for the first time the presence of two components of the cholesterol side chain cleavage system in freshly obtained granulosa cells, and provide direct evidence for the trophic effect of FSH and its presumed mediator, cyclic AMP, on the synthesis of cytochrome P-450scc in granulosa cells.     

A covalently linked complex of cytochrome P-450 with adrenodoxin. Localization of the adrenodoxin binding site of cytochrome P-450

Cytochrome P-450scc and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of a cross-linked complex and 6% of free cytochrome P-450scc was obtained after purification on cholate-Sepharose. Cytochrome P-450scc in the cross-linked complex is not reduced in the presence of NADPH and adrenodoxin reductase, but completely preserves its high spin form in the presence of Tween-20 or pregnenolone. The use of radioactive labelled adrenodoxin, chemical cleavage of cytochrome P-450scc from the cross-linked complex by o-iodosobenzoic acid and HPLC for separation of peptides demonstrated that the cytochrome P-450scc complex with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450scc, i.e., Leu 88-Trp108 and Leu368-Trp417.