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.     

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.     

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles.

Cytochrome P-450scc can be reconstituted into a phospholipid bilayer in the absence of added detergent by incubation of purified hemoprotein with preformed phosphatidylcholine vesicles. Salt effects demonstrate that the primary interaction between the cytochrome and phospholipid vesicles is hydrophobic rather than ionic; in contrast, neither adrenodoxin reductase nor adrenodoxin will bind to phosphatidylcholine vesicles by hydrophobic interactions. Insertion of cytochrome P-450scc into a phospholipid bilayer results in conversion of the optical spectrum to a low spin type, but this transition is markedly diminished if cholesterol is incorporated within the bilayer. Vesicle-reconstituted cytochrome P-450scc metabolizes cholesterol within the bilayer (turnover = 13 nmol/min/nmol of cytochrome P-450scc); virtually all (greater than 94%) of the cholesterol within the vesicle is accessible to the enzyme. "Dilution" of cholesterol within the bilayer by increasing the phospholipid/cholesterol ratio at a constant amount of cholesterol and cytochrome P-450scc results in a decreased rate of side chain cleavage, and cytochrome P-450scc incorporated into a cholesterol-free vesicle cannot metabolize cholesterol within a separate vesicle. In addition, activity of the reconstituted hemoprotein is sensitive to the fatty acid composition of the phospholipid. These results indicate that the cholesterol binding site on vesicle-reconstituted cytochrome P-450scc is in communication with the hydrophobic bilayer of the membrane. The reducibility of vesicle-reconstituted cytochrome P-450scc as well as spectrophotometric and activity titration experiments show that all of the reconstituted cytochrome P-450scc molecules possess an adrenodoxin binding site which is accessible from the exterior of the vesicle. Activity titrations with adrenodoxin reductase also demonstrate that a ternary or quaternary complex among adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc is not required for catalysis, a finding consistent with our proposed mechanism of steroidogenic electron transport in which adrenodoxin acts as a mobile electron shuttle between adrenodoxin reductase and cytochrome P-450 (Lambeth, J.D., Seybert, D.W., and Kamin, H. (1979) J. Biol. Chem. 254, 7255-7264.     

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.     

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)     

The formation of binary and ternary complexes of cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex. The implication in ACTH function.

Binary and ternary complexes of bovine adrenocortical mitochondrial cytochrome P-450scc with adrenodoxin and adrenodoxin reductase.adrenodoxin complex are formed in the presence of cholesterol and Emulgen 913. Both cholesterol and Emulgen 913 are required for the binding of cytochrome P-450scc with adrenodoxin. Since phospholipids are able to replace Emulgen 913 in this reaction, in vivo phospholipids of the mitochondrial inner membrane appear to play the function of the detergent. The dissociation constants of the cytochrome.adrenodoxin complex are 0.3 to 0.4 microM at 130 microM dimyristoylphosphatidylcholine and 0.9 microM at 120 microM Emulgen 913, whereas the dissociation constant for the ternary complex of cytochrome P-450scc with adrenodoxin reductase and adrenodoxin is 4.0 microM at 150 microM Emulgen 913. The stoichiometry of binary and ternary complexes reveals the 1:1 and 1:1:1 molar ratios, respectively, judging from chemical analyses after the fractionation of the complexes by gel filtration. Emulgen 913, Tween 20, ethylene glycol, myristoyllysophosphatidylcholine, dimyristoylphosphatidylcholine, and phosphatidylethanolamine show the enhanced activity of cholesterol side chain cleavage reaction with cytochrome P-450scc, adrenodoxin, adrenodoxin reductase, and NADPH. These results, in conjunction with earlier experiments, lead us to the proposal on the structure of the hydroxylase complex in the membrane and to the hypothesis on the regulation of the enzymatic activity by the availability of substrate cholesterol to the cytochrome. Hence, we propose a mobile P-450scc hypothesis for the response of the mitochondrion to adrenocorticotropic hormone stimuli.     

Conformational change of the heme moiety of the ferrous cytochrome P-450scc-phenyl isocyanide complex upon binding of reduced adrenodoxin

Reduction of cytochrome P-450scc(SF) (SF, substrate free) purified from bovine adrenocortical mitochondria with sodium dithionite (Na2S2O4) or with beta-NADPH mediated by catalytic amounts of adrenodoxin and adrenodoxin reductase in the presence of phenyl isocyanide produced a ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex with Soret absorbance maximum at 455 nm having a shoulder at 425 nm. On the other hand, when a preformed cytochrome P-450scc(SF)-adrenodoxin complex was reduced chemically or enzymatically under the same conditions, the absorbance spectrum showed drastic changes, i.e., an increase in intensity at 425 nm and a concomitant decrease in intensity at 455 nm. Similar spectral changes could be produced by addition of the same amount of reduced adrenodoxin afterward to the ferrous cytochrome P-450scc(SF)-phenyl isocyanide complex. Titration experiments with adrenodoxin showed that (1) a 1:1 stoichiometric saturation of the spectral change was obtained for both the absorbance increase at 425 nm and the absorbance decrease at 455 nm, (2) there was no spectral change in the presence of 0.35 M NaCl, and (3) there was no spectral change for cytochrome P-450scc(SF) whose Lys residue(s) essential to the interaction with adrenodoxin had been covalently modified with PLP. These results suggest that ternary complex formation of ferrous cytochrome P-450scc(SF)-phenyl isocyanide with reduced adrenodoxin caused a conformational change around the ferrous heme moiety. By analysis of temperature and pH dependencies of the spectral change of the ternary complex, it was suggested that this conformational change may reflect the essential step for electron transfer from reduced adrenodoxin to the ferrous-dioxygen complex of cytochrome P-450scc.     

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.     

Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).     

The side-chain cleavage of cholesterol sulfate--I. The effect of adrenodoxin on the binding of cholesterol sulfate to cytochrome P-450scc

Difference spectroscopy was used to measure the binding of cholesterol sulfate (CS) to cytochrome P-450scc. The uncomplexed cytochrome and the complex of the cytochrome with adrenodoxin (ADX) were both titrated with CS in order to test whether ADX increased the affinity of the cytochrome for the sterol sulfate. The addition of ADX to the cytochrome had different effects on the binding of the sterol sulfate depending on several factors including: (1) The method of preparation of the cytochrome P-450scc, (2) The concentration of cytochrome P-450scc, (3) The method by which CS was suspended in aqueous solution, and (4) Whether or not the solutions of cytochrome contained non-ionic detergents. The results of this study suggest that the method of isolation of cytochrome P-450scc, and non-ionic detergents, greatly modulate the apparent affinity of cytochrome P-450scc for CS. In the absence of detergents the addition of adrenodoxin to dilute solutions of cytochrome P-450scc appears to enhance only slightly (1- to 2-fold) the affinity of the cytochrome for the sterol sulfate.     

Effects of cholesterol and adrenodoxin binding on the heme moiety of cytochrome P-450scc: a resonance Raman study

The effects of cholesterol and adrenodoxin binding on resonance Raman spectra of cytochrome P-450scc in both oxidized and CO-reduced states were examined. Upon cholesterol binding, oxidized cytochrome P-450scc showed a significant shift of spin equilibrium from low-spin to high-spin state. Addition of adrenodoxin caused a complete conversion of cholesterol-bound oxidized cytochrome P-450scc to a pure high-spin state that was considered to be in the hexacoordinated state judged by the v10 mode at 1620 cm-1 and v3 mode around 1485 cm-1. Cholesterol in substrate binding site may oppose a linear and perpendicular binding of carbon monoxide to the reduced heme iron, leading to the distorted Fe-C-O linkage. This is based on the following observations: (1) an increase of the Fe-CO stretching frequency to 483 from 477 cm-1 upon addition of cholesterol; (2) an enhanced photodissociability of bound carbon monoxide of CO complex of cytochrome P-450scc in the presence of cholesterol. As another aspect of the effect of cholesterol on the CO complex form of cytochrome P-450scc, the enhanced stability of the native form ("P-450" form) was observed. There was no additional effect of reduced adrenodoxin on the Raman spectra of the CO-reduced form of cytochrome P-450scc.     

Study of the role of lysine residues of the cholesterol hydroxylating cytochrome P-450 by a method of chemical modification

Chemical modification of cytochrome P-450scc by lysine-specific reagents has been performed. Modification of the hemoprotein was shown to result in the loss of its ability to interact with adrenodoxin. With a view of identifying lysine residues involved in the interaction with adrenodoxin, cytochrome P-450scc was modified by succinic anhydride in the presence of adrenodoxin. After the removal of ferredoxin, the modification was performed with the use of a radioactively labeled reagent. Subsequent hydrolysis of the succinic hemoprotein by chymotrypsin and separation of the peptides obtained by high pressure liquid chromatography resulted in the isolation of seven chymotryptic peptides containing labeled lysine residues. These amino acid sequences were identified. The role of lysine residues of cytochrome P-450scc in complex formation with adrenodoxin is discussed.     

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.     

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.     

Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.     

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.     

A shunted system of electron transport from NAD(P)H to cholesterol-hydroxylating cytochrome P-450 in adrenal cortex mitochondria

The two main approaches presently used for cytochrome P-450scc modelling are as follows: i) the use of chemical compounds carrying activated oxygen species, e. g., peracids, organic hydroperoxides, iodosobenzene, etc., ii) the use of electrochemical reduction in the presence of redox-active compounds. In the present work, a new model system for simulation of steroidogenic electron transfer is proposed, which reduces cytochrome P-450 scc by NADPH in the absence of adrenodoxin reductase and adrenodoxin. Phenazine methosulfate is used as an electron carrier. More than 95% of cytochrome P-450scc is reduced in a model system. The reduction kinetics is characterized by a lag phase, thus indicating complex formation between cytochrome P-450scc and phenazine methosulfate or formation of intermediate reducing equivalents. NADH may also serve as an electron donor for cytochrome P-450scc. Phenazine methosulfate can reduce microsomal cytochrome P-450 LM2 and b5, but not cytochrome P-450 LM4. Superoxide dismutase does not affect the reduction, thus indicating that O9.- is not involved in the reduction process. The mechanism of hemoprotein reduction and the nature of intermediates which can be formed in the model system is proposed.     

Regulation of rat adrenal messenger RNA and protein levels for cytochrome P-450s and adrenodoxin by dietary sodium depletion or potassium intake

The effect of low sodium and high potassium intake on rat adrenal zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR) were studied during a 7-day period, by analyzing mRNA and protein levels of various enzymes involved in aldosterone synthesis. In ZG significant increases in cytochrome P-450scc, P-450c21, P-450(11 beta), adrenodoxin mRNA and protein levels were observed after 2 days with either diet, and at day 7 these levels were further increased. The largest mRNA induction was observed at day 7 in sodium-depleted rats for P-450(11 beta), with a 4-fold increase, followed by 2.7- and 2.0-fold increases for P-450scc and P-450c21, respectively. A pattern similar to those of P-450scc and P-450(11 beta) was observed for adrenodoxin with a 2.1-fold increase after 7 days of Na+ restriction. In K(+)-loaded rats mRNA levels for P-450scc, P-450(11 beta), P-450c21, and adrenodoxin were also increased by 2.2-, 2.1-, 1.5-, and 1.9-fold respectively. Protein levels of these enzymes were also measured in ZG and showed increases similar to those of their respective mRNAs for both treatments. On the other hand, mRNA levels of P-450scc, P-450(11 beta), P-450c21, and adrenodoxin in ZFR were found significantly lower than in ZG, although they were slightly increased for both treated groups of rats as compared with controls. In addition, ZFR protein levels of corresponding enzymes did not fluctuate significantly under both ionic regimens. In conclusion, both low sodium and high potassium intakes act primarily on ZG. Their action on plasma aldosterone seems to be mediated by increasing both mRNA and protein and levels of steroidogenic enzymes, especially at the early step (cytochrome P-450scc) and even more at the late steps (cytochrome P-450(11 beta]. In addition, a close relationship appears to exist between the two mitochondrial P-450s and their electron donor adrenodoxin, since their mRNA and protein levels were similarly enhanced for both diets used.     

Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane

Selective chemical modification of adrenocortical cytochrome P-450scc, responsible for key stages of steroid biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450scc tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450scc inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450scc results in changes in the hemeprotein spectral properties and its conformation which indicates to the involvement of essential tyrosine residue(s) in the heme-protein interaction. Cholesterol and adrenodoxin (high-spin effectors) prevent the inactivation of cytochrome P-450scc with tetranitromethane, i.e., protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450scc molecule are discussed.     

Stoichiometry of mitochondrial cytochromes P-450, adrenodoxin and adrenodoxin reductase in adrenal cortex and corpus luteum. Implications for membrane organization and gene regulation

We have estimated the concentrations of cytochromes P-450scc and P-45011 beta and the electron-transfer proteins adrenodoxin reductase and adrenodoxin in the adrenal cortex and corpus luteum using specific antibodies against these enzymes. While in the adrenal cortex the concentrations of these enzymes are relatively constant in different animals and show no significant sex differences, in corpora lutea they vary considerably and can increase at least up to fifty-fold over the levels found in the ovary. The average relative concentrations of adrenodoxin reductase, adrenodoxin and P-450 are 1:3:8 in the adrenal cortex (which has two cytochromes P-450, P-450scc and P-450(11) beta, in equal concentrations) and 1:2.5:3 in the corpus luteum (which has only P-450scc). We further present evidence that the levels of cytochrome c oxidase also show a degree of correlation with the levels of the mitochondrial steroidogenic enzymes.