The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-, 18-hydroxylation, and aldehyde synthesis

In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum.     

Aldosterone biosynthesis and cytochrome P-45011 beta: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-45011 beta 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 11 beta-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-45011 beta. 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-45011 beta 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.     

Adaptation of aldosterone biosynthesis to sodium and potassium intake in the rat

The steroidogenic response of rat adrenal zona glomerulosa to stimulators is variable and depends on the activity of biosynthetic steps involved in the conversion of deoxycorticosterone (DOC) to aldosterone (Aldo). Corticosterone methyl oxidations (CMO) 1 and 2 are stimulated by sodium restriction and suppressed by potassium restriction. These slow alterations are accompanied by the appearance or disappearance of a specific zona glomerulosa mitochondrial protein with a molecular weight of 49,000. Induction of CMO 1 and 2 activities and the appearance of the 49 K protein can also be elicited in vitro by culture of rat zone glomerulosa cells in a medium with a high potassium concentration. The 49 K protein crossreacts with a monoclonal antibody raised against purified bovine adrenal cytochrome P-450(11 beta). The same antibody stains a protein with a molecular weight of 51,000 in rat zona fasciculata mitochondria and in zone glomerulosa mitochondria of rats in which CMO 1 and 2 activities have been suppressed by potassium restriction and sodium loading. The 51 K crossreactive protein was purified to electrophoretic homogeneity by chromatography on octyl-sepharose. In a reconstituted enzyme system, it converted DOC to corticosterone (B) and to 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) but not to 18-hydroxycorticosterone (18-OH-B) or Aldo. A partially purified 49 K protein preparation from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen converted DOC to B, 18-OH-DOC, 18-OH-B and Aldo. According to these results, rat adrenal cytochrome P-450(11 beta) exists in two different forms, with both of them capable of hydroxylating DOC in either the 11 beta- of the 18-position, but with only the 49 K form capable of catalyzing CMO 1 and 2. The adaptation of aldosterone biosynthesis to sodium deficiency or potassium intake in rats is due to the appearance of the 49 K form of the enzyme in zona glomerulosa mitochondria.     

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.     

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.     

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-45011 beta 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-45011 beta, 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.     

Purification and characterization of two distinct forms of rat adrenal cytochrome P450(11) beta: functional and structural aspects

It is generally accepted that the last three steps of aldosterone biosynthesis are catalyzed by a single enzyme, i.e., cytochrome P450(11) beta (P450XIB). We have previously reported that rat adrenal mitochondria may be capable of producing two forms of P450(11) beta which differ in molecular weight (49 and 51 kDa). In the present study we describe the purification, the enzymatic activities, and some structural properties of these two proteins. Using zona fasciculata mitochondria, the 51-kDa protein was purified to electrophoretic homogeneity by means of octyl-Sepharose chromatography. In a reconstituted system the protein catalyzed 18- and 11 beta-hydroxylation of deoxycorticosterone, but exhibited no 18-hydroxylation or 18-hydroxydehydrogenation of corticosterone. The 49-kDa protein was isolated from zona glomerulosa mitochondria of rats kept on a low-sodium, high-potassium regimen. Using octyl-Sepharose chromatography, it could be separated from the 51-kDa protein. A reconstituted eluate fraction, containing the 49-kDa protein, converted deoxycorticosterone not only to 18-OH-deoxycorticosterone and corticosterone, but also to 18-OH-corticosterone and aldosterone. These findings indicate that the rat adrenal cortex is capable of producing two distinct forms of active cytochrome P450(11) beta. A structural relationship of the 49- and 51-kDa proteins was indicated by experiments involving limited proteolysis. Thus, digestion with alpha-chymotrypsin and V8-protease yielded very similar peptide maps for both proteins. During potassium repletion of potassium-deficient rats, the disappearance of the active 51-kDa protein coincided with the appearance of the 49-kDa protein. These results are suggestive of a post-translational processing mechanism converting the 51-kDa protein into the smaller 49-kDa form. However, the 49-kDa protein might also be encoded by a distinct gene, regulated separately depending on the physiological conditions.     

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).     

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 binding of cortisol to adrenal mitochondria

Binding of tritiated cortisol to adrenal zona glomerulosa mitochondria was studied and compared with that of corticosterone. Cortisol was shown to bind specifically to the inner membrane of zona glomerulosa mitochondria. Corticosterone and cortisol had similar apparent association constants (Ka) and concentrations of binding sites. The methodology was validated by obtaining similar Ka from both binding plots and kinetic data. Cortisol binding was inhibited by pretreatment with sodium dithionite, and displaced by deoxycorticosterone, corticosterone, 18-hydroxy-corticosterone, 11 beta-hydroxy-18-ethynyl-progesterone and metyrapone, but not by cholesterol. These results suggest that cortisol and corticosterone bind to the same cytochrome P-450.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

Isolation of two distinct cytochromes P-45011 beta with aldosterone synthase activity from bovine adrenocortical mitochondria

Two distinct forms of cytochrome P-45011 beta, with apparent molecular weights of 48,500 (48.5K) and 49,500 (49.5K), have been isolated from bovine adrenocortical mitochondria. Their amino acid sequences up to the 19th position from the N-terminus were only different at the 6th position (Val and Ala for the 48.5K and 49.5K enzymes, respectively). Each sequence was assignable to a distinct cDNA clone for cytochrome P-450(11) beta (Kirita, S., et al. [1988] J. Biochem. 104, 683-686), indicating that the two proteins originate from different genes in bovine adrenocortical cells. Both forms of cytochrome P-450(11) beta were capable of catalyzing aldosterone synthesis as well as the 11 beta- and 18-hydroxylation of 11-deoxycorticosterone. Thus, at least two distinct cytochrome P-450(11) beta species exist in the adrenal cortex and participate in steroidogenesis.     

Sodium depletion of rats induces a mitochondrial protein in the zona glomerulosa of the adrenal cortex

Sodium restriction of rats resulted in a marked increase in the concentration of a mitochondrial protein with a mol. wt of 49000 in the zona glomerulosa of the adrenal cortex. The same protein had been previously induced in potassium deficient rats by potassium repletion. Since both sodium depletion and potassium repletion are known to stimulate the activity of the mitochondrial mixed function oxidase involved in the conversion of corticosterone to aldosterone, the present observation supports the hypothesis that the potassium-induced protein might represent cytochrome P-450CMO (corticosterone methyl oxidase).     

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.     

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

We have recently reported that bovine adrenocortical cytochrome P-45011 beta catalyzes 19-hydroxylation of 18-hydroxy-11-deoxycorticosterone (18(OH)DOC) in addition to 11 beta-hydroxylation of the steroid. In this report, we examine the presence of these two activities in 18(OH)DOC and 11 beta- 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 11 beta-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 11 beta-hydroxylation of 18(OH)DOC well occurred in these species. The last group comprising rat had very low activity both of 11 beta- and 19-hydroxylations when 18(OH)DOC was used as the substrate, whereas both 11 beta- 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.     

Mitochondrial steroid metabolism in the inner and outer zones of the guinea-pig adrenal cortex

Previous investigations have demonstrated that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea-pig adrenal cortex produce far more cortisol than those from the inner zone (zona reticularis). Studies were carried out to compare mitochondrial steroid metabolism in the two zones. Protein and cytochrome P-450 concentrations were similar in outer and inner zone mitochondria. However, the rate of 11 beta-hydroxylation was significantly greater in the outer zone despite the fact that substrates for 11 beta-hydroxylation (11-deoxycortisol, 11-deoxycorticosterone) produced larger type I spectral changes in inner zone mitochondria. The apparent affinities of 11-deoxycortisol and 11-deoxycorticosterone for mitochondrial cytochrome(s) P-450 were similar in the two zones. In both inner and outer zone mitochondria, 11 beta-hydroxylation was inhibited by metyrapone but unaffected by aminoglutethimide. Cholesterol sidechain cleavage activity, measured as the rate of conversion of endogenous cholesterol to pregnenolone, was far greater in outer than inner zone mitochondria. Addition of exogenous cholesterol or 25-hydroxycholesterol to the mitochondrial preparations did not affect pregnenolone production in either zone. Addition of pregnenolone to outer zone mitochondria produced a reverse type I spectral change (delta A 420-390 nm), suggesting displacement of endogenous cholesterol from cytochrome P-450. In inner zone mitochondria, pregnenolone induced a difference spectrum (delta A 425-410 nm) similar to the reduced vs oxidized cytochrome b5 spectrum. A b5-like cytochrome was found to be present in the mitochondrial preparations. Prior reduction of the cytochrome with NADH eliminated the pregnenolone-induced spectral change in inner zone mitochondria but had no effect in outer zone preparations. The results suggest that differences in mitochondrial steroid metabolism between the inner and outer adrenocortical zones account in part for the differences in cortisol production by cells in each zone.     

18-Hydroxy-11-deoxycortisol: a new steroid isolated from incubations of the adrenal with 11-deoxycortisol

Cortisol has been shown to be metabolized in the zona glomerulosa of the adrenal gland through the same pathway involving the cytochrome P-450, corticosterone methyl oxidase by which corticosterone is transformed to 18-hydroxycorticosterone and aldosterone. When cortisol is the precursor, 18-hydroxycortisol and 18-oxocortisol are formed. 18-Hydroxycortisol can also be made at a similar rate in the bovine zona fasciculata and reticularis as in the zona glomerulosa. We studied the possibility that the formation of 18-hydroxycortisol in the zona fasciculata and reticularis might be through a different pathway involving initial 18-hydroxylation of 11-deoxycortisol before 11 beta-hydroxylation. Rat adrenal capsules or cores were incubated with 10 micrograms of cortisol or 11-deoxycortisol and the formation of 18-hydroxycortisol was measured by radioimmunoassay. Both capsules and cores transformed 11-deoxycortisol to 18-hydroxycortisol, but cortisol was only transformed in the capsular portion. Sixty-two rat adrenals were incubated with 10 mg of 11-deoxycortisol and the putative steroid, 18-hydroxy-11-deoxycortisol, was purified by TLC and HPLC and subjected to gas chromatography mass spectrometry. The mass spectra indicated that the steroid isolated was indeed 18-hydroxy-11-deoxycortisol. The function of this steroid is still unknown.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including microsomes, described above, and mitochondria. The amount of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40–50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20–30 sec of incubation with deoxycholate.The content of RNA, phospholipids, and cytochromeb ₅ in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.     

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.