Adrenocortical Cytochrome P-450 Research Articles

123I]Iodometomidate for Molecular Imaging of Adrenocortical Cytochrome P450 Family 11B Enzymes

Due to advances in conventional imaging, adrenal tumors are detected with increasing frequency. However, conventional imaging provides only limited information on the origin of these lesions, which represent a wide range of different pathological entities. New specific imaging methods would therefore be of great clinical value. We, therefore, studied the potential of iodometomidate (IMTO) as tracer for molecular imaging of cytochrome P450 family 11B (Cyp11B) enzymes. Inhibition of Cyp11B1 and Cyp11B2 by IMTO, etomidate, metomidate, and fluoroetomidate was investigated in NCI-h295 cells and in Y1 cells stably expressing hsCyp11B1 or hsCyp11B2. Pharmacokinetics and biodistribution after iv injection of [(123/125)I]IMTO were analyzed in mice in biodistribution experiments and by small-animal single-photon emission computed tomography (SPECT). Furthermore, four patients with known adrenal tumors (two metastatic adrenal adenocarcinomas, one bilateral adrenocortical adenoma, and one melanoma metastasis) were investigated with [(123)I]iodometomidate-SPECT. In cell culture experiments, all compounds potently inhibited both Cyp11B1 and Cyp11B2. Adrenals showed high and specific uptake of [(123/125)I]IMTO and were excellently visualized in mice. In patients, adrenocortical tissue showed high and specific tracer uptake in both primary tumor and metastases with short investigation time and low radiation exposure, whereas the non-adrenocortical tumor did not exhibit any tracer uptake. We have successfully completed the development of an in vivo detection system of adrenal Cyp11B enzymes by [(123)I]IMTO scintigraphy in both experimental animals and humans. Our findings suggest that [(123)I]IMTO is a highly specific radiotracer for imaging of adrenocortical tissue. Due to the general availability of SPECT technology, we anticipate that [(123)I]IMTO scintigraphy may become a widely used tool to characterize adrenal lesions.

Photoaffinity labeling of cytochrome [formula omitted] with methyltrienolone as a probe for the substrate binding region

Methyltrienolone, a synthetic steroid, was used as a photoaffinity ligand for steroid-binding proteins. The enzymatic activity of bovine adrenocortical cytochrome P-450 11β was inhibited by methyltrienolone in a competitive manner without exposure to light and cytochrome P-450 11β was photolabeled with methyltrienolone after irradiation with UV light. The addition of 11-deoxycorticosterone during photolabeling protected cytochrome P-450 11β from photolabeling. Photolabeled cytochrome P-450 11β was digested with TPCK-treated trypsin and the peptide fragments were separated with a reverse-phase HPLC system. The labeled peptide was analyzed and its amino acid sequence was determined to be Trp 428-Leu 429-Asp 430-Arg 431. Alignment of the primary structure of cytochrome P-450 11β with that of cytochrome P-450 cam revealed that the identified sequence corresponds to the region between the β3-sheet and L-helix of cytochrome P-450 cam . This region of mammalian cytochromes P-450 shows poor homology with that of cytochrome P-450 cam , but is well-conserved, especially at Trp-428 and preceding amino acids, as the aromatic region. The present results demonstrate that the labeled sequence contributes in part to the formation of the substrate binding pocket of cytochrome P-450 11β which was not expected from the results of the primary sequence alignment with cytochrome P-450 cam .

Dynamic structures of adrenocortical cytochrome P-450 in proteoliposomes and microsomes: protein rotation study

Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the immunohistochemical and biochemical localization of the cytochrome P-450 scc-linked monooxygenase system in the adult rat brain

Immunohistochemical and biochemical studies were performed on the brains of adult female and male rats using a specific antibody against bovine adrenocortical cytochrome P-450 scc. The results showed that in both male and female rats, the myelinated regions of the white matter are selectively immunostained throughout the brain and that even in rats pretreated with colchicine, there is never positive staining of neuronal cell bodies and their dendrites in any brain region. Western immunoblotting with the P-450 scc antibody and enzymatic assays revealed that P-450 scc and cholesterol side-chain cleavage activity were present in a homogenate derived from the cortical white matter, but not detectable in that from the cerebral cortex. Furthermore, quantitation of the P-450 scc protein in the immunoblots indicated that the concentration of P-450 scc in the cortical white matter of both female and male rat brains is approx. 3–4 pmol per mg tissue protein. Thus it could be concluded that in the adult rat brain, P-450 scc and cholesterol side-chain cleavage activity are selectivity localized only in the myelinated region of the white matter.

Modified acetylenic steroids as potent mechanism-based inhibitors of cytochrome P-450 SCC

Synthesized 20-(4-tetrahydropyranyl-1-butynyloxy)-5-pregnen-3α,20β-diol [steroid I] and 20-(3-tetrahydropyranyl-1-propargyloxy)-5-pregnen-3α,20β-diol [steroid III] have been found to inactivate purified adrenocortical cytochrome P-450 SCC. When incubated with the enzyme under turnover conditions, steroid I inactivated cytochrome P-450 SCC by about 85% in 40 min. This is in contrast to the free triol analog, steroid II which inactivated the enzyme by only 45% within the same incubation period. A comparison of steroid III with its free triol analog, steroid IV, also showed that the diol is a more effective inactivator of the enzyme than the triol. The partition ratio was calculated by two different methods. Each of the steroids I–IV bound to the enzyme with spectrophotometric dissociation constant ( K s ) in the micromolar range, producing Type II low spin spectra changes during titration of the enzyme. In addition, it was found that the binding of each of the compounds to the enzyme occurred without inactivation of the enzyme and that the inactivation under turnover condition, is not as a result of conversion to the denatured P-420 species. This demonstrated that steroids I and III could correctly be designated as mechanism-based (suicide) inhibitors. The kinetic studies demonstrated that steroids with the tetrahydropyranyl substituent are more potent inhibitors of cytochrome P-450 SCC as shown by an initial turnover rate of 0.06 min −1, an inactivation rate constant of 0.05 min −1, and a partition ratio of about 1.0 for steroid I. Based on our finding, possible mechanisms of inactivation of cytochrome P-450 SCC by these acetylenic steroids are propsoed.

Lipid regulation of bovine cytochrome P450 11β activity

Purified bovine adrenocortical cytochrome P450 11β has been reconstituted into phospholipid vesicles using a detergent dialysis procedure. Using this reconstituted system, we have examined the effect of changes in the fatty acyl substituents of the lipids on the catalytic activity of the enzyme. The studies reported here show that cytochrome P450 11β exhibits a completely different response to changes in the fatty acyl groups from that shown by cytochrome P450 scc. Cytochrome P450 11β displays maximal activity in lipid vesicles composed of saturated lipids, such as dipalmitoyl and dimyristoyl phosphatidylcholines, with turnover numbers ranging from 35 to 60 min −1. Incremental increases of phospholipids such as diphytanoyl and dioleoyl phosphatidylcholines result in a progressive inhibition of 11β hydroxylase activity; most of this kinetic effect is attributable to a significant decrease in V max accompanied by modest changes in K m for the steroid substrate deoxycorticosterone. Diphosphatidyl glycerol (cardiolipin), which has been previously shown to activate cytochrome P450 scc, is a potent inhibitor of the 11β hydroxylase activity of cytochrome P450 11β, with half maximum inhibition observed in vesicles containing 4–5 mol% diphosphatidyl glycerol. Kinetic analysis demonstrates that this inhibition by diphosphatidyl glycerol is reflected in both a decrease in V max and relatively large increases (up to sevenfold) in K m for the steroid substrate. These effects on the 11β hydroxylase activity may have important implications for the in vivo regulation of not only the 11β hydroxylase activity, but also the other catalytic activities of this enzyme, particularly 18- and 19-hydroxylase and oxidase activities.

Sensitive assay of cytochrome P450 scc activity by high-performance liquid chromatography

We have developed a simple procedure for analyzing the reaction intermediates and product of the cholesterol side-chain cleavage system by high-performance liquid chromatography with uv absorption monitoring. After the cholesterol side-chain cleavage system had been incubated and the reaction then halted by heat treatment, the product was converted into 3-one-4-en steroid showing intense absorption at 240 nm upon reaction with cholesterol oxidase. The converted steroids were then analyzed by normal-phase HPLC. In consequence, the catalytic activity of the reconstituted adrenocortical cytochrome P450 scc system was readily assayed with a sensitivity more than 10-fold higher by this conversion. Also, it was shown that 22 R-hydroxycholest-4-en-3-one could serve as a good substrate for cytochrome P450 scc and that the 20 R,22 R-dihydroxy derivative could be clearly detected as a reaction intermediate in the reconstituted system.

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-450 11 β catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-450 11 β , whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.

Rapid spectral scan and stopped-flow studies of carbon monoxide binding to bovine adrenocortical cytochrome P-450 scc

Carbon monoxide binding with both cholesterol-free (low-spin) and cholesterol-bound (high-spin) reduced forms of purified cytochrome P-450 scc has been investigated by rapid-scan and stopped-flow spectrometry. CO binding occurs within 150 ms at 25°C for both forms of P-450 scc, with a typical absorption maximum at 450 nm. Isosbestic points occur at the following wavelengths: between reduced-CO and reduced cholesterol-free P-450 scc at 434 and 471 nm; between reduced-CO and reduced cholesterol-bound P-450 sec at 433 and 469 nm. Both the ‘on’ ( k 1) and ‘off’ rate constants ( k −1) are found to be independent of pH between pH 5 and 9. The mean values of k 1 for cholesterol-free ((1.8 ± 0.2) · 10 5 M −1 · s −1) and cholesterol-bound (((1.9 ± 0.1) · 10 5 M −1 · s −1) P-450 scc are almost identical, while the mean value of k −1 for the former (((2.3 ± 0.3) · 10 s −1) is about double that of the latter ((1.2 ± 0.1) · 10 s −1). This suggests the instability of the reduced-CO complex in the absence of cholesterol.

Molecular organization (topography) of cytochrome P-450 11 β in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis

Cytochrome P-450 11 β from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450 11 β following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11β hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450 11 β is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450 11 β on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.

Bovine adrenocortical cytochrome P-450(17 alpha). Regulation of gene expression by ACTH and elucidation of primary sequence.

In the steroidogenic pathway of the adrenal cortex, 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha)) is a major regulatory enzyme. Previous studies have shown that stimulation of 17 alpha-hydroxylase activity occurs upon treatment of bovine adrenocortical cells in culture with adrenocorticotropic hormone (ACTH), via cAMP, due to an increase in the enzyme concentration. Alterations in the levels of this enzyme result in pronounced changes in the pattern of steroids produced, with the potent glucocorticoid cortisol, as well as the sex steroids, being products of 17 alpha-hydroxylated steroid precursors. In the present study, the identification and sequencing of two cloned DNA molecules, pB17 alpha-1 and pcD17 alpha-2, which are complementary to mRNA sequences encoding bovine adrenal cortex P-450(17 alpha), are reported. Clone pcD17 alpha-2 contains an open reading frame coding for the complete amino acid sequence of P-450(17 alpha) which consists of 509 amino acids and has a molecular weight of 57,251. By comparison to other forms of cytochrome P-450, we conclude that P-450(17 alpha) is a member of a previously unidentified family within the P-450 multigene superfamily. Single bovine and human mRNA species of approximately equal to 1850 bases in length hybridize to these clones. Regulation of the concentration of this RNA has been studied using bovine adrenocortical cells in culture. Treatment with ACTH or dibutyryl cAMP causes an increase in the content of P-450(17 alpha) RNA in as little as 2 h, and its concentration increases greater than 20-fold by 8 h. In contrast, other steroidogenic enzymes that have been studied respond more slowly to ACTH. Actinomycin D and cycloheximide block induction of P-450(17 alpha) RNA, indicating that regulation of 17 alpha-hydroxylase activity is controlled at the level of transcription and requires ongoing protein synthesis.

Monoclonal antibodies against bovine adrenal cytochrome P-450 11β and cytochrome P-450 scc. Their isolation, characterization and application to immunohistochemical analysis of adrenal cortex

Seven monoclonal antibodies directed against bovine adrenocortical cytochrome P-450 11β were isolated. They were found to be immunoglobulins each having distinct affinity to the antigen. Five clones had IgGI heavy chains, whereas the other two had IgG2b heavy chains. All the clones had k light chains. All the monoclonal antibodies recognized a protein among mitochondrial proteins of bovine adrenal cortex, whose mol. wt, 50,000, was the same as that of cytochrome P-450 11β. Among the monoclonal antibodies isolated, monoclonal antibody 258 recognized a protein of Mr 50,000 in rat adrenal mitochondria, a protein of Mr 47,000 in pig adrenocortical mitochondria, a protein of Mr 50,000 in guinea pig adrenal mitochondria, a protein of Mr 55,000 in rabbit adrenal mitochondria and a protein of Mr 50,000 in human adrenocortical mitochondria. These results suggest that each of these proteins, having a mol. wt around 50,000, recognized by monoclonal antibody 258 is very likely to be the cytochrome P-450 11β of adrenocortical mitochondria of each animal species. Monoclonal antibody 42718 inhibited by 50% the steroid hydroxylation activity of cytochrome P-450 11β. In addition to the monoclonal antibodies against cytochrome P-450 11β, three monoclonal antibodies directed against bovine adrenocortical cytochrome P-450 scc were also isolated and characterized. Immunohistochemical staining of bovine adrenal cortex with the use of these two kinds of monoclonal antibodies revealed that the contents of both cytochrome P-450 11β and cytochrome P-450 scc were greater in the zona fasciculata and zona reticularis than in the zona glomerulosa. Electron microscopical observation of immunoperoxidase-stained preparations confirmed the presence of these cytochromes in the mitochondria.

Cross-linking studies of adrenocortical cytochrome P-450 scc. 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-450 scc. 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-450 scc with adrenodoxin has been obtained by using dimethyl-3,3′-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450 scc 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-450 scc. 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-450 scc. This is consistent with the model mechanism of electron transfer in the organized complex.

19-hydroxylation of 18-hydroxy-11-deoxycorticosterone by adrenal mitochondria prepared from various animal species

We have recently reported that bovine adrenocortical cytochrome P-450 11β catalyzes 19-hydroxylation of 18-hydroxy-11-deoxycorticosterone (18(OH)DOC) in addition to 11β-hydroxylation of the steroid. In this report, we examine the presence of these two activities in 18(OH)DOC and 11β-and 18-hydroxylation activities on deoxycorticosterone (DOC) among the adrenal mitochondria prepared from man, ox, pig, rabbit, guinea-pig and rat. The results indicate that these animals could be classified into three groups with respect of these hydroxylation activities. Mitochondria of the first group comprising ox and pig showed rather high 19- and 11β -hydroxylation activities on 18(OH)DOC compared to the hydroxylation activities on DOC. Mitochondria prepared from the second group which comprised rabbit, guinea-pig and man showed low 19-hydroxylation activity on 18(OH)DOC, whereas the 11β -hydroxylation of 18(OH)DOC well occurred in these species. The last group comprising rat had very low activity both of 11β- and 19-hydroxylations when 18(OH)DOC was used as the substrate, whereas both 11β- and 18-hydroxylations of DOC were high in rat adrenal mitochondria. No significant difference of these activities could be found between zona glomerulosa cells and zonae fasciculata-reticularis cells of bovine adrenal cortex, and between adrenal mitochondria from spontaneously hypertensive rat and those from WKY normotensive rat.

Adrenocortical cytochrome P-450 responsible for cholesterol side chain cleavage (P-450 scc) is phosphorylated by the calcium-activated, phospholipid-sensitive protein kinase (protein kinase C)

Purified bovine adrenocortical cytochrome P-450 scc (specific for cholesterol side chain cleavage in the inner mitochondrial membrane) was selectively phosphorylated in vitro by a Ca 2+-activated, phospholipid-sensitive protein kinase (protein kinase C) preparation, whereas cyclic AMP dependent and two cyclic nucleotide independent kinases were ineffective. Cytochrome P-450 scc incorporated a maximum of 4 mol of phosphate in the presence of protein kinase C within 15 min at 30°C, with apparent Km and Vmax of 0.14 μmol and 0.76 pmol/min, respectively. Serine and threonine were the two target aminoacids phosphorylated in a ratio of about 1:1. In the presence of 1 μM Ca 2+, a mixture of phosphatidylserine and diolein (or a potent tumor promoter phorbol ester) was required for optimal cytochrome P-450 scc phosphorylation. In addition, purified inner mitochondrial membrane preparations from adrenocortical mitochondria were found to contain protein kinase C activity. These findings, together with the previous demonstration that activators of protein kinase C such as a potent phorbol ester activates steroidogenesis of intact adrenocortical cells, suggest that phosphorylation of P-450 scc should be examined for its possible role in the regulation of adrenocortical functions.

Chemical modification of adrenocortical cytochrome P-450 see with tetranitromethane

Selective chemical modification of adrenocortical cytochrome P-450 see, responsible for key stages of steroi biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450 see tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450 see inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450 see 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-450 see with tetranitromethane, i.e. protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450 see molecule are discussed.

The activity and biosynthesis of cholesterol side-chain cleavage enzyme in cultured immature pig testis cells

An IgG fraction from antiserum raised against cholesterol side-chain cleavage cytochronie P-450 (cytochrome P-450 SCC) purified from bovine adrenocortical mitochondria cross-reacted with immature pig testis cytochrome P-450 SCC in an Ouchterlony double diffusion system. This property of the IgG fraction was utilized for the immunoisolation of cytochrome P-450 SCC of immature pig testis cells in culture. The molecular weight of the immunoisolate from [ 35S]methionine-labeled pig testis Leydig cells was similar to that of purified bovine adrenocortical cytochrome P-450 SCC (49000). The testis iron-sulfur protein similarly immunoisolated using an IgG fraction from antiserum raised against adrenodoxin purified from bovine adrenocortical mitochondria was also of molecular weight similar to that of adrenodoxin (13000). The rates of synthesis of cytochrome P-450 SCC and testis iron-sulfur protein in immature pig testis cells in culture, when incubated with hCG (100 mU/ml) for 48 h, were 3- and 6-fold greater, respectively, than those in cells incubated in the absence of hCG. This result is suggestive that the synthesis of cytochrome P-450 SCC and testis iron-sulfur protein of immature pig testis tissue is stimulated by gonadotropin.

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

Organ specificities of cytochrome P-450 s21 (steroid 21-hydroxylase) were investigated. The substrate specificity of the liver cytochrome P-450 s21 was found to be different from that of adrenocortical cytochrome P-450 s21. The steroid 21-hydroxylase activity of liver microsomes decreased with treatment by sodium phenobarbital or β-naphthoflavone and was inhibited by anti-cytochrome b 5 immunoglobulin, although that of adrenocortical microsomes was not. The liver cytochrome P-450 s21 was immunochemically similar to adrenocortial cytochrome P-450 s21. The bovine liver and adrenocortical cytochromes P-450 s21 showed immunoprecipitin lines against antibody to bovine adrenocortical cytochrome P-450 s21. The molecular masses of the bovine liver and adrenocortical cytochromes P-450 s21 were 60 ± 1 and 50 ± 1 kDa, respectively. The content of cytochrome P-450 s21 and its percentage for the total microsomal cytochrome P-450 were immunochemically determined using the antibody to the bovine adrenocortical cytochrome P-450 s21.

Spectroscopic evidence for the formation of a transient species during cytochrome P-450 SCC induced hydroperoxysterol-glycol conversions

Spectroscopic analysis of the interaction of the epimeric 20-hydroperoxy derivatives of cholesterol with bovine adrenocortical cytochrome P-450 SCC preparations suggested the formation of a transient species. The intermediate was detected at 4°C and characterized by a minimum at 412 nm in the difference spectrum.

Integration of purified adrenocortical cytochrome P-450 11β into phospholipid vesicles

Cytochrome P-450 supporting steroid 11β hydroxylase activity (cyt P-450 11β) was purified from bovine adrenal cortex mitochondria using a procedure, which included an octyl-sepharose adsorption step and elution of the protein in the presence of phosphatidyl-choline. Purified cyt P-450 11β could then be included into phosphatidyl choline-phosphatidyl ethanolamine (1 : 1) spherical vesicles (20–50 nm in diameter) during their formation upon gel filtration, as demonstrated by the protein refractoriness to trypsin hydrolysis. After inclusion into the phospholipid vesicles, cyt P-450 11β remained stable and expressed full 11β hydroxylase activity in a reconstituted system including purified adrenodoxin and adrenodoxin reductase.