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

18and 1 lp-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 18and 1 lp-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 18and 1 lp-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18and 1 lp-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18and llp-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 18and 1 lphydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18and 1 lp-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 18and 1 lphydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited llp-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18and llp-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 18and 1 lp-hydroxylation of deoxycorticosterone is discussed. Supplementary key words cholesterol side chain cleavage * mass fragmentography In a previous work, 18and 1 lp-hydroxylation of different steroids in mitochondria and reconstituted systems from rat and bovine adrenals were studied (1). It was found that 18and 1 lp-hydroxylase activities with deoxycorticosterone as substrate were unchanged under various assay conditions (suboptimal amounts of substrate, cytochrome P-450, adrenodoxin and/or adrenodoxin reductase). The ratio also remained constant during inhibition with carbon monoxide and metyrapone. From these results, it was suggested that identical or at least very similar types of cytochrome P-450 are involved in 18and 1 lp-hydroxylation of deoxycorticosterone. In the present work, we have by different means further investigated the possibility that a single enzyme system is involved in 18and 1 lp-hydroxylation of deoxycorticosterone. Treatment of rats with potassium chloride in drinking fluid has been reported to increase 18-hydroxylation of corticosterone (2, 3). It was considered to be of interest to determine whether this increase in 18-hydroxylase activity is followed by a similar increase in 1 lp-hydroxylase activity. Long-term treatment with ACTH is known to have different effects on different hydroxylations in the adrenals (4). It was also considered to be of interest to study if 1 lpand 18-hydroxylation and cholesterol side chain cleavage are affected differently by such treatment. In some recent work, methods have been described for preparation of fractions of cytochrome P-450 from bovine adrenals that either have a high cholesterol 592 Journal of Lipid Research Volume 18, 1977 by gest, on Jauary 5, 2018 w w w .j.org D ow nladed fom side chain cleavage activity and a low 1 lp-hydroxylase activity or vice versa (5-8). 18-Hydroxylase activity has not been followed through such purifications. The relatively low 18-hydroxylase activity of bovine adrenal cytochrome P-450 make such studies difficult (cf. 1). Rat adrenal cytochrome P-450 has a relatively high ratio between 18-hydroxylase activity and 1 lp-hydroxylase activity (cf. l), and in the present work we have therefore tried to subject rat adrenal cytochrome P-450 to partial purification and have studied the ratio between 18and 1 lp-hydroxylation of deoxycorticosterone during the procedures. In previous work from this laboratory ( l ) , it was shown that a crude mitochondrial system as well as a reconstituted cytochrome P-450 system catalyzed both 18and 1 lp-hydroxylation of deoxycorticosterone but only 1 lp-hydroxylation of progesterone. Unlabeled progesterone was found to inhibit both 18and 1 lphydroxylation of tritium-labeled deoxycorticosterone, and deoxycorticosterone inhibited 1 lp-hydroxylation of [4-14C]progesterone. In the present work, attempts have been made to determine whether or not these inhibitions are of a competitive nature. Inhibition of a competitive nature would suggest that the same binding site or at least very similar binding sites are involved in 1 lp-hydroxylation of progesterone and deoxycorticosterone and in 18-hydroxylation of deoxycorticosterone. MATERIALS AND METHODS [1,2-3H2]Corticosterone (sp act 40 pCi/pmol) was purchased from New England Nuclear (Boston, MA). [ 1 ,2-3Hz]Deoxycortic~~terone (sp act 10 pCi/pmol), [4-14C]progesterone (sp act 3 pCi/pmol), [4-14C]cholesterol (sp act 59-60 pCi/pmol), and [4-14C]pregnenolone (sp act 59-60 pCi/pmol) were purchased from Radiochemical Centre (Amersham, England). The purity of the labeled compounds was ascertained by means of thin-layer chromatography followed by radio-scanning of the chromatoplates (cf. below). Aldosterone, corticosterone, d oxycorticosterone, and ACTH were obtained from Sigma Chemical Co. (St. Louis, MO). 18-Hydroxycorticosterone was purchased from Steraloids (Pawling, NY), Sephadex G-100 and G-200 were obtained from Pharmacia (Uppsala, Sweden). DEAE-Cellulose was obtained from Whatman (Maidstone, England). Assay of cytochrome P-450 and NADPHcytochrome P-450 reductase activity Cytochrome P-450 was assayed from the absorbance of the carbon monoxide-cytochrome P-450 complex, TABLE 1. Amount of insoluble cytochrome P-450 and cholesterol in submitochondrial preparations from rat adrenals Cytochrome Preparation Protein P-450 Cholesterol