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.     

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

Highly purified cytochrome P-450 11 beta-/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-11 beta-hydroxylase (IgG) before assay. It is concluded that cytochrome P-450 11 beta-/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.     

Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.     

Aldosterone biosynthesis by a reconstituted cytochrome P-45011 beta system

[3H]Corticosterone was incubated with cytochrome P-45011 beta 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-45011 beta, 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.     

Cloning and expression of a cDNA for human cytochrome P-450aldo as related to primary aldosteronism

A cDNA clone encoding human aldosterone synthase cytochrome P-450 (P-450aldo) has been isolated from a cDNA library derived from human adrenal tumor of a patient suffering from primary aldosteronism. The insert of the clone contains an open reading frame encoding a protein of 503 amino acid residues together with a 3 bp 5'-untranslated region and a 1424 bp 3'-untranslated region to which a poly(A) tract is attached. The nucleotide sequence of P-450aldo cDNA is 93% identical to that of P-450(11) beta cDNA. Catalytic functions of these two P-450s expressed in COS-7 cells are very similar in that both enzymes catalyze the formation of corticosterone and 18-hydroxy-11-deoxycorticosterone using 11-deoxycorticosterone as a substrate. However, they are distinctly different from each other in that P-450aldo preferentially catalyzes the conversion of 11-deoxycorticosterone to aldosterone via corticosterone and 18-hydroxycorticosterone while P-450(11)beta substantially fails to catalyze the reaction to form aldosterone. These results suggest that P-450aldo is a variant of P-450(11)beta, but this enzyme is a different gene product possibly playing a major role in the synthesis of aldosterone at least in a patient suffering from primary aldosteronism.     

The reoxidation of cytochrome P-450 by paraquat inhibits aldosterone biosynthesis from 18-hydroxycorticosterone

Paraquat is an artificial electron carrier that captures electrons from reduced cytochrome P-450 instead of the natural acceptors, thus decreasing the concentration of reduced mitochondrial cytochrome P-450. In the present study, paraquat inhibited the biosynthesis of aldosterone from 18-hydroxycorticosterone by mitochondria from duck adult adrenal gland, under aerobic conditions. Since paraquat did not induce any change in the absorption spectrum of highly purified cytochrome P-450 11 beta, the possibility of a displacement of steroid by the drug is ruled out. Moreover, paraquat did not affect oxidative phosphorylating chain nor did it alter by itself the chemical structure of 18-hydroxycorticosterone. In our conditions, the inhibitory role of paraquat seems restricted to a capture of electrons from reduced cytochrome P-450. Under the same conditions metopirone and spironolactone, known to bind cytochrome P-450 11 beta at the steroid binding site, also inhibited the reaction. Altogether these results show that for aldosterone synthesis from 18-hydroxycorticosterone to take place, the steroid binding site on cytochrome P-450 must be accessible to 18-hydroxycorticosterone and that the cytochrome P-450 must be the direct donor of reducing equivalents. Hence, cytochrome P-450 appears as the final linking point between 18-hydroxycorticosterone and the reducing equivalents provided by NADPH.     

Adrenal P-450scc modulates activity of P-45011 beta in liposomal and mitochondrial membranes. Implication of P-450scc in zone specificity of aldosterone biosynthesis in bovine adrenal.

Bovine adrenal P-45011 beta catalyzes the 11 beta- and 18-hydroxylation of corticosteroids as well as aldosterone synthesis. These activities of P-45011 beta were found to be modulated by another mitochondrial cytochrome P-450 species, P-450scc. The presence together of P-45011 beta and P-450scc in liposomal membranes was found to remarkably stimulate the 11 beta-hydroxylase activity of P-45011 beta and also stimulate the cholesterol desmolase activity of P-450scc. The stimulative effect of P-450scc on 11 beta-hydroxylase activity diminished by the addition of protein-free liposomes to proteoliposomes containing P-45011 beta and P-450scc, thus showing P-450scc to interact with P-45011 beta in the same membranes. Kinetic analysis of this effect indicated the formation of an equimolar complex between P-45011 beta and P-450scc on liposomal membranes. P-45011 beta in the complex had not only stimulated activity for 11 beta- and 18-hydroxylation of 11-deoxycorticosterone but also suppressed activity for production of 18-hydroxycorticosterone and aldosterone. When the inner mitochondrial membranes of zona fasciculata-reticularis from bovine adrenal were treated with anti-P-450scc IgG, aldosterone formation was stimulated to a greater extent than that of zona glomerulosa. This indicates the aldosterone synthesizing activity of P-45011 beta in the zona fasciculata-reticularis to be suppressed by interaction with P-450scc. The zone-specific aldosterone synthesis of P-45011 beta in bovine adrenal may possibly be induced by differences in interactions with P-450scc of mitochondrial membranes in each zone.     

Reconstruction and study of a multi-enzyme system by 11 beta-hydroxylase steroids

The reconstitution of the steroid 11 beta-hydroxylase system based on the homogeneous proteins isolated from bovine adrenocortical mitochondria, cytochrome P-450 (P-450 (11 beta), 19-20.5 nmol of heme P-450 per 1 mg of protein), adrenodoxin (Adx) and adrenodoxin reductase (AR) was carried out. The reconstitution of the multienzyme system requires the presence of a non-ionic detergent due to the high hydrophobicity of P-450 (11 beta). Low concentrations of Tween 20 (below 0.015% or 115 microM) stimulate the reaction of steroid 11 beta-hydroxylation by improving the hemoprotein solubility. With a further increase in the detergent concentration, the reaction is inhibited due to the inactivation of the cytochrome and its impaired interaction with Adx. The electron transfer activity of adrenodoxin reductase and the dienzyme AR-Adx complex does not change within the Tween 20 concentration range of 0-0.4%. In solutions with the optimal concentration of Tween 20 (0.010-0.015%), the concentrations of AR and Adx providing for the half-maximum hydroxylation activity are 9 nM for AR and 280 nM for Adx. It was shown that in a reconstituted 11 beta-hydroxylase system, 75% of the reducing equivalents are involved in the formation of oxygen radicals, whereas 25%--in hydroxylation. 74% of the radical species are, in their turn, formed in the active site of the hemoprotein, while 26%--in the Fe2S2 center of adrenodoxin. The radical formation process predominates over the 11 beta-hydroxylation within a wide range of Adx/cytochrome ratios, i.e., 1.0-100. The hydroxylation substrate induces a 4-fold increase in the electron transfer rate by stimulating the enzymatic reduction of P-450 (11 beta), but only 35% of the additional reduced equivalents are consumed by the 11 beta-hydroxylation and 65%--by the oxygen radical formation.     

Enzymatic activities of P-450(11 beta)s expressed by two cDNAs in COS-7 cells

Expression plasmids were constructed using two cDNA clones of P-450(11 beta), pcP-450-(11 beta)-2, and pcP-450(11 beta)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11 beta) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11 beta)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11 beta)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11 beta)-3 was greater than that by P-450(11 beta)-2.     

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.     

Adrenal cortical 11 beta-hydroxylase and side-chain cleavage enzymes. Requirement for the A- or B-pyridyl ring in metyrapone for inhibition

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.     

Molecular cloning and expression of cDNAS encoding rat aldosterone synthase: variants of cytochrome P-450(11 beta)

Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-450(11 beta),aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-450(11 beta),aldo-2) had an extension peptide identical to that of rat P-450(11 beta). Glu at the 320th position of P-450(11 beta),aldo-1 was replaced with Lys in P-450(11 beta),aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-450(11 beta). Constructed expression vector containing the cDNA for extension peptide of P-450(11 beta) and the mature protein of P-450(11 beta),aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.     

The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation.

We have utilized 11beta-hydroxylase activity and visible absorption spectrophotometry to detect possible complex formation among adrenodoxin reductase, adrenodoxin, and cytochrome P-450(11)beta. At low ionic strength, a 1:1 complex between adrenodoxin reductase and adrenodoxin occurs but does not support maximal rates of 11beta hydroxylation; at least 1 additional molecule of adrenodoxin in excess of the 1:1 complex is required for full hydroxylase activity. Spectrophotometric titration of a mixture of adrenodoxin reductase and cytochrome P-450(11)beta with adrenodoxin indicates sequential formation of 1:1 complexes between adrenodoxin reductase and adrenodoxin and then between a second adrenodoxin and cytochrome P-450(11beta; the adrenodoxin-cytochrome P-450(11)beta complex is only detectable when the concentration of adrenodoxin exceeds that of adrenodoxin reductase.     

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.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.     

Functional expression of the cDNAs encoding rat 11 beta-hydroxylase [cytochrome P450(11 beta)] and aldosterone synthase [cytochrome P450(11 beta, aldo)]

Expression plasmids containing two cDNAs of a rat cytochrome P450(11 beta) family, pcP450(11 beta)-62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21-26] and pcP450(11 beta, aldo)-46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245-252], were constructed and introduced into COS-7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11 beta) and P450(11 beta, aldo) were determined by using 11-deoxycorticosterone, corticosterone, 18-hydroxy-11-deoxycorticosterone, 18-hydroxycorticosterone, or 19-hydroxy-11-deoxycorticosterone as a substrate. Cytochrome P450(11 beta) catalyzed 11 beta-, 18- and 19-hydroxylations of 11-deoxycorticosterone and 19-oxidation or 19-hydroxy-11-deoxycorticosterone at substantial rates, 18-hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11 beta, aldo) catalyzed 11 beta- and 18-hydroxylations of 11-deoxycorticosterone, 18-hydroxylation of corticosterone and aldosterone production from 11-deoxycorticosterone or corticosterone. But neither 19-hydroxylation of 11-deoxycorticosterone nor 19-oxidation of 19-hydroxy-11-deoxycorticosterone was catalyzed by cytochrome P450(11 beta, aldo).     

Common characteristics of the cytochrome P-450 system involved in 18- and 11 beta-hydroxylation of deoxycorticosterone in rat adrenals.

18- and 11beta-Hydroxylation of deoxycorticosterone and side chain cleavage of cholesterol were studied in mitochondria and submitochondrial reconstituted systems prepared from rat and bovine adrenals. A mass fragmentographic technique was used that allows determination of hydroxylation of both exogenous and endogenous cholesterol. The following results were obtained. (1) Treatment of rats with excess potassium chloride in drinking fluid increased mitochondrial cytochrome P-450 as well as 18- and 11beta-hydroxylase activity in the adrenals. Cholesterol side chain cleavage was not affected. In the presence of excess adrenodoxin and adrenodoxin reductase, cytochrome P-450 isolated from potassium chloride-treated rats had higher 18- and 11beta-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18- and 11beta-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18- and 11beta-hydroxylase activity. The amount of cytochrome P-450 in the adrenals was not affected by the treatment. It was shown with isolated mitochondrial cytochrome P-450 in the presence of excess adrenodoxin and adrenodoxin reductase that the effect of ACTH was due to increase of side chain cleavage activity per nmol cytochrome P-450. Side chain cleavage of exogenous cholesterol was affected more than that of endogenous cholesterol. (3) Gel chromatography of soluble cytochrome P-450 prepared from rat and bovine adrenal mitochondria yielded chromatographic fractions having either a high 18- and 11beta-hydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18- and 11beta-hydroxylase activity was approximately constant, provided the origin of cytochrome P-450 was the same. (4) Addition of progesterone to incubations of deoxycorticosterone with soluble or insoluble rat adrenal cytochrome P-450 competitively inhibited 18- and 11beta-hydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited 11beta-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18- and 11beta-hydroxylation have similar properties and that the binding site for deoxycorticosterone is similar or identical in the two hydroxylations. The possibility that the same specific type of cytochrome P-450 is responsible for both 18- and 11beta-hydroxylation of deoxycorticosterone is discussed.     

Potassium intake and aldosterone biosynthesis: the role of cytochrome P-450

K+ Repletion for 48 h of rats previously kept on a low K+ diet for 2 weeks specifically increased the conversion of corticosterone into aldosterone and 18-hydroxycorticosterone by incubated capsular fractions of rat adrenal tissue. This increase in the activity of the final steps of aldosterone biosynthesis was not accompanied by an increase in capsular adrenal mitochondrial cytochrome P-450 concentration. By contrast, an increased corticosterone-induced absorbance change (BI) was consistently found in capsular adrenal mitochondria upon K+ repletion. In addition, a type I-like absorbance change was induced with 18-hydroxy-11-deoxycorticosterone but not with 18-hydroxycorticosterone. Therefore, K+ repletion of K+ depleted rats specifically increased the binding of corticosterone and possibly 18-hydroxy-11-deoxycorticosterone to the 18-methyl oxidase enzyme complex. Whether this increased binding was due to an increase in enzyme protein concentration or due to a better availability of the substrate to the enzyme, could not be decided from these experiments.     

The final step of aldosterone biosynthesis is catalyzed by an NADPH-dependent and molecular oxygen-requiring enzyme

Using sonicated mitochondria fraction prepared from bovine adrenal glomerulosa cells, aldosterone biosynthesis from 18-hydroxycorticosterone was examined as its final step, as production of [³H]-aldosterone from [³H]-corticosterone was strongly reduced by addition of non-radioactive 18-hydroxycorticosterone during the incubation. Significant conversion of 18-hydroxycorticosterone to aldosterone by the mitochondria sonicate was observed in the presence of NADPH, but not NADP⁺. This reaction was almost completely inhibited in the atmosphere of 100% carbon monoxide in the presence of either NADP⁺, or NAD⁺, and significantly reduced in the mixture of carbon monoxide and oxygen (90:10) in the presence of NADPH. Several drugs, such as SU compounds, spironolactone, amphenone B and SKF 525A which affect cytochrome P-450 blocked production of aldosterone from 18-hydroxycorticosterone. From these results, we conclude that a mixed function oxidase involving a cytochrome P-450 is engaged in the final course of aldosterone biosynthesis.     

Hydroxylation of steroids in a dienzyme system consisting of cytochrome P-450 and immobilized adrenodoxin

Experimental systems for the hydroxylation of steroids (11-deoxycorticosterone and cholesterol) with reduced electron transfer chain, in which flavoprotein was omitted, were investigated. Incubation of chemically reduced immobilized adrenodoxin either with cytochrome P-45011 beta or cytochrome P-450scc in the presence of substrate of hydroxylation and oxygen yields the specific reaction products, corticosterone or pregnenolone. The catalytic activity of the experimental dienzyme systems proves the possibility of the steroid hydroxylation mechanism based exclusively on dissociation and reassociation of the electron transporting protein complexes.