Cytochrome P-45011β Research Articles

Conversion of 11-deoxycorticosterone and corticosterone to aldosterone by cytochrome P-450 11β-/18-hydroxylase from porcine adrenal

Highly purified cytochrome P-450 11β-/18-hydroxylase and the electron carriers adrenodoxin and adrenodoxin reductase were prepared from porcine adrenal. When the enzyme was incubated with the electron carriers, 11-deoxycorticosterone (DOC) and NADPH, the following products were isolated and measured by HPLC: corticosterone, 18-hydroxy-11-deoxycorticosterone (18-hydroxyDOC), 18-hydroxycorticosterone and aldosterone. All of the DOC consumed by the enzyme can be accounted for by the formation of these four steroids. Aldosterone was identified by mass spectroscopy and by preparing [ 3H] aldosterone from [ 3H]corticosterone followed by recrystallization at constant specific activity after addition of authentic aldosterone. Corticosterone and 18-hydroxycorticosterone were also converted to aldosterone. Conversion of corticosterone and 18-hydroxycorticosterone to aldosterone required P-450, both electron carriers, NADPH and substrate. The reaction is inhibited by CO and metyrapone. Moreover, all three activities of the purified enzyme decline at the same rate when the enzyme is kept at room temperature for various periods of time and when the enzyme is treated with increasing concentrations of anti-11β-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11β-/18-hydroxylase can convert DOC to aldosterone via corticosterone and 18-hydroxycorticosterone. The stoichiometry of this conversion was found to be 3 moles of NADPH, 3 moles of H + and 3 moles of oxygen per mole of aldosterone produced: C 21 H 30 O 3 + 3 NADPH + 3 H + + 3 O 2 → C 21 H 28 O 5 + 3 NADP + + 4 H 2 O

Gerbil adrenal 11β- and 19-hydroxylating activities respond similarly to inhibitory or stimulatory agents: Two activities of a single enzyme

A high level of steroid 19-hydroxylation is exhibited by adrenal mitochondria of the gerbil, Meriones, unguiculatus, that accounts for the ability of that species to produce nearly equal amounts of corticosterone and 19-hydroxycorticosterone ( Proc. Soc. exp. Biol. Med. 165 (1980) 69–74). Inhibitors of steroidogenesis and a polyclonal antibody against bovine cytochrome P-450 11β were used to determine if the agents would effect differential or parallel suppression of 19- vs 11β-hydroxylation by gerbil adrenal mitochondria in vitro. The inhibitors (0.1–60 μM) tested (listed in order of decreasing effectiveness) were imazalil, metyrapone, miconazole and 4-hydroxyandrostenedione. With each inhibitor the degree of suppression of 11β-hydroxylation was accompanied by a parallel decline in 19-hydroxylation. The addition of the polyclonal antibody preparation also produced equivalent declines in the rates of the two hydroxylation reactions. The addition of ACTH 1μM to primary cultures of gerbil adrenal cells brought about nearly equal increases in the secretion of 11β- and 19-hydroxylated steroids into the culture media. These results support the hypothesis that the 11β-hydroxylase of gerbil adrenal mitochondria has the capacity to carry out 11β- and 19-hydroxylations with nearly equal facility.

Levels of adrenodoxin, NADPH-cytochrome P-450 reductase and cytochromes P-450 11β, P-450 21, P-450 scc, in adrenal zona fasciculata-reticularis tissue from androgen-treated rats

Treatment of rats with methylandrostenediol (MAD), an anabolic androgen, caused a profound reduction (65%) in the level of cytochrome P-450 11 β in rat adrenocortical mitochondria as measured by immunoblots using a specific antibody. The decreases in mitochondrial cytochrome P-450 scc (15%) and adrenodoxin (20%) were much less than that observed for cytochrome P-450 11 β . A 35% decrease in adrenal microsomal cytochrome P-450 21 and NADPH-cytochrome P-450 reductase levels was brought about by the treatment with MAD. The data establish that the preferential decrease in adrenal steroid 11β-hydroxylase activity associated with androgen treatment results from a decrease in cytochrome P-450 11 β . This is consistent with the role of 11-deoxycorticosterone in the pathogenesis of androgen-induced hypertension in rats.

Molecular cloning and nucleotide sequence of DNA of mitochondrial cytochrome P-450(11 beta) of bovine adrenal cortex.

cDNA clones of the mRNA for bovine adrenal cytochrome P-450(11 beta) were isolated. Sequence analysis of a 4 kb long cDNA revealed the primary structure of P-450(11 beta), which consisted of 503 amino acids (Mr: 57,924) and contained an extension peptide of 24 amino acids at the NH2-terminus of the mature P-450(11 beta). molecule. A bovine genomic DNA containing the 1st exon and its leader sequence of P-450(11 beta) gene was also isolated from a bovine gene library. Determination of the transcription initiation site by S1 nuclease analysis using the cloned genomic DNA confirmed that the methionine codon near the 5' side of the 4 kb long cDNA was the initiation codon. Comparisons of the primary structures among P-450(11 beta) and other forms of cytochrome P-450 including P-450(SCC) indicated that the two mitochondrial P-450s, P-450(11 beta) and P-450(SCC), were significantly different from microsomal forms of cytochrome P-450. The homology between P-450(11 beta) and P-450(SCC) was 36%, which is higher than the values between P-450(11 beta) and various microsomal P-450s. An alignment of P-450(11 beta) and P-450(SCC) to give maximum matching showed four highly conserved regions (C-1, C-2, C-3, and C-4). The homology values of these regions were 58-70%, considerably higher than the overall homology between these two mitochondrial P-450s. A putative heme binding site and a steroid binding site were located in the conserved regions. Hydropathy profiles of P-450(11 beta) and P-450(SCC) were very similar. A definite difference was noticed at the NH2-terminal portion between mitochondrial and microsomal types of P-450. Microsomal type of cytochrome P-450 had a hydrophobic sequence consisting of about 20 amino acids, whereas mitochondrial type had an extension peptide containing many positively changed amino acids.

Biphasic 19-nor-deoxycorticosterone excretion after adrenal enucleation is associated with altered adrenal mitochondrial cytochrome P450 11 beta-, 18-, and 19-hydroxylase activities.

After adrenal enucleation (AE) rats avidly retain sodium (early phase), but after 7-10 days they lose this sodium avidity (late phase). Although increased production of a mineralocorticoid, 19-nor-deoxycorticosterone (19-Nor-DOC), has been implicated, 19-Nor-DOC levels during the early and late phases of AE have not been systematically measured. Furthermore it is not known why 19-Nor-DOC production should increase during a time when production of 11 beta- and 18-hydroxylated corticosteroids are decreased in AE. The purpose of this study was to examine the 11 beta, 18-, and 19-hydroxylase pathways in the early and late phases of AE. The results demonstrate increased urinary 19-Nor-DOC and decreased 18-OH-DOC and corticosterone excretion in the early phase of AE at a time when adrenal mitochondrial 11 beta- and 18-hydroxylase activities were decreased but 19-hydroxylase activity was unchanged. During the late phase of AE, urinary 19-Nor-DOC had decreased and 18-OH-DOC and corticosterone had increased to levels indistinguishable from those in sham controls. This reduction in 19-Nor-DOC was associated with a decrease in 19-hydroxylase activity in AE. Since the 11 beta, 18-, and 19-hydroxylases have a common substrate (DOC), it is possible that differential flux of DOC through these pathways could account for the changes in steroid production in AE. These data suggest that the increased 19-Nor-DOC excretion in AE may be due to alterations in enzyme activity leading to a shunting of DOC into the 19-Nor-DOC pathway. In addition, the synchronicity of 19-Nor-DOC with sodium excretion suggests that it has an important role in the pathogenesis of the sodium retention in AE.

Aldosterone biosynthesis and cytochrome P-450 11β: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-450 11β has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11β-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K. protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-450 11β. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-450 11β in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.

Production of 19-hydroxy-11-deoxycorticosterone and 19-oxo-11-deoxycorticosterone from 11-deoxycorticosterone by cytochrome P-450 11β

Incubation of 11-deoxycorticosterone with a cytochrome P-450 11β-reconstituted system yielded, in addition to corticosterone and 18-hydroxy-11-deoxycorticosterone, a new steroid product. The retention time of the new product was identical with that of authentic 19-hydroxy-11-deoxycorticosterone on high performance liquid chromatography (HPLC). The turnover number of 19-hydroxy-11-deoxycorticosterone formation was 7.0 mol/min/mol P-450. When a large amount of cytochrome P-450 11β was used for the reaction and the products were analyzed by HPLC, the 19-hydroxy-11-deoxycorticosterone peak disappeared from the chromatogram and concomitantly new unidentified peaks appeared. These results suggest that 19-hydroxy-11-deoxycorticosterone was further metabolized to other steroids by cytochrome P-450 11β. Therefore, we next incubated 19-hydroxy-11-deoxycorticosterone with cytochrome P-450 11β and analyzed the reaction products by HPLC. The above-mentioned unidentified peaks appeared again in the chromatogram. The retention time of one of the peaks coincided with that of authentic 19-oxo-11-deoxycorticosterone. This peak substance was purified by repeated HPLC and subjected to mass spectrometry and 1H NMR analyses. Its field desorption mass spectrum (FD-MS) showed a M + peak at m/e 344. The 1H NMR spectrum showed the signal of an aldehyde proton instead of those of hydroxymethyl protons at the C-19 position. These results suggest that cytochrome P-450 11β can catalyze the 19-hydroxylation of 11-deoxycorticosterone, and the 19-hydroxy-11-deoxycorticosterone produced is further oxidized at the C-19 position to 19-oxo-11-deoxycorticosterone.

Effect of calmodulin on aldosterone synthesis by a cytochrome P-45011 beta-reconstituted system from bovine adrenocortical mitochondria.

Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta.

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.

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.

Synthesis of aldosterone by a reconstituted system of cytochrome P-45011 beta from bovine adrenocortical mitochondria.

When corticosterone was incubated with cytochrome P-45011 beta purified from bovine adrenocortical mitochondria in the presence of adrenodoxin, NADPH-adrenodoxin reductase and an NADPH generating system, aldosterone as well as 18-hydroxycorticosterone were formed with turnover numbers of 0.23 and 1.1 nmol/min/nmol P-450, respectively. Phospholipids extracted from adrenocortical mitochondria remarkably enhanced the activity of aldosterone formation by the cytochrome P-45011 beta-reconstituted system. The apparent Km and turnover number were estimated to be 6.9 microM and 2.0 nmol/min/nmol P-450 for aldosterone formation in the presence of the lipidic extract. When 18-hydroxycorticosterone was tested as a substrate, cytochrome P-45011 beta showed catalytic activity for aldosterone synthesis with an apparent Km and turnover number of 325 microM and 5.3 nmol/min/nmol P-450, respectively. Carbon monoxide and metyrapone inhibited the production of aldosterone from corticosterone and that from 18-hydroxycorticosterone. These results suggest that conversion of corticosterone and of 18-hydroxycorticosterone to aldosterone occurs through P-45011 beta-catalyzed reaction.

Mode of action of butylated hydroxyanisole (BHA) and other phenols in preventing loss of 11β-hydroxylase activity in cultured bovine adrenocortical cells

When cultured bovine adrenocortical cells are incubated with cortisol, or other steroids that are pseudosubstrates for 11β-hydroxylase (cytochrome P-450 11β), the activity of the enzyme decreases. In previous experiments, three substances were shown to protect 11β-hydroxylase against loss of enzymatic activity in the presence of pseudosubstrates: BHA (butylated hydroxyanisole, 2(3)- tert-butyl-4-methoxyphenol), dimethyl sulfoxide (DMSO), and metyrapone. The present experiments examine the protective effects of several phenolic analogs of BHA in this system, and compare their activities to that of DMSO and metyrapone. When a variety of analogs of BHA were tested for their abilities to prevent loss of 11β-hydroxylase activity in cultured adrenocortical cells incubated with 50 μM cortisol for 24 hr, phenol itself was found to be about equipotent with BHA. Addition of methyl, methoxy and benzyl groups to phenol did not diminish protective activity of the compound, but addition of one and particularly two tert-butyl groups greatly diminished activity. Thus, BHT (2,6-di- t-butyl-4-methylphenol) was inactive, in contrast to BHA. The hydroxy group of phenol was essential since benzene and fluorobenzene were inactive. Compounds with multiple hydroxyl groups were not as active as phenol itself, with the exception of catechol. No products of phenol formed during incubations of cells with cortisol were detected by high performance liquid chromatography. Estimated ec 50 values for protection of 11β-hydroxylase by phenols were about 100 μM, whereas the ec 50 values for dimethyl sulfoxide and metyrapone were 10 mM and 300 nM respectively. On a semilogarothmic plot, the dose-response curves for all these compounds were approximately parallel. To aid in determining the mechanism of protection of 11β-hydroxylase, phenols and DMSO were tested for prevention of loss of 11β-hydroxylase activity at three different oxygen concentrations (2,5, and 19% O 2). Lowering the oxygen concentration itself resulted in a small diminution of the loss of 11β-hydroxylase. Phenols and dimethyl sulfoxide were more effective at low oxygen and less effective in air. Because the cytochrome P-450 inhibitor metyrapone was found previously to be very effective in protecting 11β-hydroxylase against loss of activity, we examined whether phenols and dimethyl sulfoxide may act by directly inhibiting 11β-hydroxylase activity. In a 1-hr incubation with cells, BHA, phenol, and dimethyl sulfoxide all inhibited 11β-hydroxylase, but at concentrations that ranged from 4- to >100-fold higher than those required for protection. In contrast, for metyrapone, the ec 50 values for protection and inhibition were very similar. These results indicate that it is unlikely that phenols act simply as inhibitors but may need to bind close to the active site of the enzyme. The observed synergism with lowered oxygen suggests an involvement, in the loss of 11β-hydroxylase activity, of oxygen-centered radicals that may be reactants for protective phenols.

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.

Aldosterone biosynthesis by a reconstituted cytochrome P-450 11β system

[ 3H]Corticosterone was incubated with cytochrome P-450 11β purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [ 3H]18-hydroxycorticosterone was incubated with cytochrome P-450 11β, the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.

Zonal distribution of cytochrome P-450s (P-450 11β and P-450 scc) and their relation to steroidogenesis in bovine adrenal cortex

Zona glomerulosa and zonae fasciculata-reticularis of bovine adrenal cortex were separated by our improved method using a remodeled microtome. A considerable amount of fresh mitochondria and microsomes was quickly obtained. Specific content of cytochrome P-450 in the mitochondria was higher in zonae fasciculata-reticularis than in zona glomerulosa. When metyrapone was added to the mitochondria and the drug-induced difference spectra were measured, there appeared two phases in the titration curve; the first phase was saturated at a concentration of 3 μM and the second phase appeared at concentrations higher than 10 μM. The first phase seemed to be due to the substrate-free P-450 11β and the second phase to the cholesterol-bound P-450 scc. From these spectral studies contents of cytochrome P-450 scc and P-450 11β were estimated, and those were found to be equal for each zone. From similar studies on substrate-induced difference spectra, there seemed to be no difference between two zones in the affinity of cytochrome P-450 11β for deoxycorticosterone and 11-deoxycortisol. This was also the case with 11β-hydroxylase activity and substrate specificity for deoxycorticosterone and 11-deoxycortisol.

The relationship between NADPH-dependent lipid peroxidation and degradation of cytochrome P-450 in adrenal cortex mitochondria

The relationship between NADPH-dependent lipid peroxidation and the degradation of cytochrome P-450 has been studied in bovine adrenal cortex mitochondria. Malondialdehyde formation is accompanied by a corresponding decrease in total cytochrome P-450 content. Inhibitors of lipid peroxidation also prevent the loss of cytochrome P-450, further demonstrating a direct relationship between NADPH-dependent lipid peroxidation and degradation of P-450. To differentiate between cytochrome P-450 11β and P-450 scc, steroidinduced difference spectra were used to evaluate P-450 degradation. These measurements provide the first evidence that both P-450's are degraded during NADP-dependent lipid peroxidation with P-450 11β being much more susceptible to this process.

18,19-Dihydroxydeoxycorticosterone; A novel product of cytochrome P-450 11β-catalyzed reaction

After incubating 18-hydroxydeoxycorticosterone (18-OH-DOC) with cytochrome P-450 11β in the reconstituted system, the products were analyzed with HPLC. There appeared two product-peaks on the chromatogram, one of which was identified as a peak of 18-hydroxycorticosterone (18-OH-B), an expected product of the 11β-hydroxylation. Another peak did not coincide with those of any known corticoids. This unidentified product was further purified, and the purified material was analyzed by gas chromatography-mass spectrometry ( GC MS ). The mass spectrum showed that the unidentified product is one of the structural isomers of 18-OH-B. A further analysis with 1H-NMR spectrometry indicated that a proton resonance peak of 19-CH 3 in 18-OH-DOC disappeared in the product and the methyl group of the substrate seemed to be converted to -CH 2OH. These results suggested that the unidentified product generated from 18-OH-DOC by P-450 11β-linked hydroxylase system may be 18,19-dihydroxydeoxycorticosterone (18,19,21-trihydroxypregn-4-ene-3,20-dione; 18,19-diOH-DOC), a hitherto unreported corticoid.

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.

Purification and reconstitution of the steroid 21-hydroxylase system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical microsomes

The steroid 21-hydroxylase system was purified from bovine adrenocortical microsomes. The physicochemical properties of the cytochrome P-450 s21 were studied. The properties of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) of bovine adrenocortical microsomes have been reported previously (Hiwatashi, A. and Ichikawa, Y. (1979) Biochim. Biophys. Acta 580, 44–63). The steroid 21-hydroxylase system was reconstituted by cytochrome P-450 s21 and NADPH-cytochrome P-450 reductase in the presence of detergent. The substrate specificity of the cytochrome P-450 s21 was examined with the reconstituted system. The enzyme system was active in the 21-hydroxylations of 17 α- hydroxyprogesterone and progesterone, and the N-demethylation of (+)-benzphetamine. The cytochrome P-450 s21 purified to as high as 16–17 nmol per mg protein was an electrophoretically pure glycoprotein. The molecular weight of the cytochrome P-450 s21 was estimated to be 47 500 by SDS-polyacrylamide gel electrophoresis. Although cytochrome P-450 scc or cytochrome P-450 11β of bovine adrenocortical mitochondria did not react with antibody to cytochrome P-450 BPA of bovine liver microsomes, the cytochrome P-450 s21 formed an immunoprecipitin line against the antibody to cytochrome P-450 BPA in the Ouchterlony double diffusion test.

Comparative immunochemical studies of cytochrome P-450CAM of Pseudomonas putida and of cytochrome P-450SCC of bovine adrenocortical mitochondria.

Antibodies raised in rabbits against highly purified preparations of two cytochromes (cytochrome P‐450CAM from Pseudomonas putida and cytochrome P‐450scc from bovine adrenocortical mitochondria) were found to be specific for their respective antigens in Ouchterlony doublediffusion assays. In each case a single sharp curve was observed and no response was seen with other P‐450 hemeproteins. In enzyme inhibition studies with reconstituted monooxygenase systems both the precipitating antibodies and soluble, monovalent Fab−1 fragments obtained from them by peptic digestion were effective inhibitors. In contrast to the Ouchterlony assay, anti(P‐450CAM) not only stopped camphor hydroxylation completely but also caused measurable inhibition of the cholesterol side‐chain cleavage (desmolase) reaction while anti(P‐450scc) inhibited camphor hydrolxylation significantly in addition to abolishing desmolase activity. Iodination of cytochromes P‐450CAM and P‐450scc with the help of chloramine‐T produced soluble, radiolabeled preparations with specific activities ranging from 3 × 108 to 1 × 109 counts × min−1×μmol−1 which showed undiminished enzymatic activities and gave a single precipitin curve each in the Ouchterlony assay with anti(P‐450CAM) and anti(P‐450scc), respectively. In the iodinated preparations 60–70% of the tyrosine residues were found to be present as monoiodotyrosines while no other amino acid was labeled. A sensitive radioimmunoassay was developed, measuring inhibition of binding between 125I‐labeled cytochrome P‐450CAM and its antibodies by cross‐reacting material, which revealed substantial cross‐reactivity of cytochromes P‐45011β (88%), P‐450scc (64%) and P‐450LM‐2 (60%). Conversely, competitive binding o.125I‐labeled cytochrome P‐450sccand unlabeled cross‐reacting antigens to anti(P‐450scc demonstrated cross‐reactivities for cytochromes P‐450sccβ (60%), P‐450CAM (75%) and P‐450LM2 (90%). Incubation of cytochrome P‐450scc with adrenodoxin, and of cytochrome P‐450CAM with putidaredoxin, prior to interaction with anti(P‐450scc) and anti(P‐450CAM), respectively, caused marked decreases in the ability of the corresponding P‐450 hemeproteins to bind to their antibodies. Even the BrCN‐derived hemepeptides of cytochromes P‐450CAM and P‐450ssc were still able to recognize the antibodies elicited by their parent proteins as evidenced by significant crossreactivities in the radioimmunoassay. It is concluded that the four P‐450 hemeproteins tested in this investigation, cytochromes P‐450CAM, P‐450ssc, P‐45011β, and P‐450LM‐2, have one or more antigenic determinants in common and that a major antibody binding site is associated with the hemepeptide.