Specificity of the cholesterol side-chain cleavage enzyme system of adrenal cortex

Incubation of lanosta-8, 24-dien-3β-o1-1,2- ³H and lanost-8-en-3β-o1-1, 2-³H with an adrenocortical bovine mitochondrial acetone-dried preparation did not yield any significant ( < 0.01%) 3β-hydroxy-4, 4, 14-trimethyl-5α-pregn-8-en-20-one. Under the same conditions cholesterol-1,2-³H yielded 8.3% pregnenolone. Incubation of (20S?) — 17α, 20-dihydroxycholesterol-7-³ H yielded 0.6 to 1.6% (20SS?, 22R?) — 17α, 20, 22-trihydroxycholesterol, 1.0 to 3.2% of 17α-hydroxypregnenolone, but no significant ( < 0.02%) (20S, 22S)-17α, 20, 22-trihydroxycholesterol. In another experiment incubation of cholesterol-1, 2-³H yielded 5% pregnenolone, 0.5% 17α-hydroxypregnenolone, 0.2% (20R?,22R?)-20, 22-dihydroxy-cholesterol, but no significant ( < 0.01%) 17α-hydroxy-cholesterol, (20S?) -17α, 20-dihydroxycholesterol or (20S?, 22R?)-17α, 20, 22-trihydroxycholesterol.     

Stimulation of cholesterol side-chain cleavage enzyme activity by cAMP and hCG in MA-10 Leydig tumor cells

Using a cloned Leydig tumor cell line (designated MA-10), we have studied the activity of cholesterol side-chain (CSCC) enzyme, the rate-determining step in steroidogenesis, in mitochondria isolated from cells pretreated either with human chorionic gonadotropin (hCG) or dibutyryl cyclic adenosine monophosphate (dbcAMP). Results showed a slight but significant increase in CSCC activity with treatment by cAMP (25% increase) and hCG (60% increase), as compared to mitochondria isolated from nontreated control cells. However, this stimulation of CSCC activity appears to be of limited significance when compared to the approximately 1000-fold or greater increase observed in progesterone production in the presence of hCG or dbcAMP. On the other hand, unstimulated MA-10 cells or isolated mitochondria efficiently converted 25-hydroxycholesterol and 22R-hydroxycholesterol into progesterone, and this conversion was not affected by cycloheximide. The addition of cholesterol to intact cells or to isolated mitochondria did not affect progesterone production. Our observations clearly indicate that given the proper hydroxy substrates (22R-hydroxycholesterol or 25-hydroxycholesterol), MA-10 Leydig cells are able to convert them into progesterone without any stimulation by steroidogenic stimuli, i.e. cAMP or hCG. Since MA-10 Leydig cells can efficiently convert 22R-hydroxycholesterol--an intermediate in CSCC reaction--into progesterone, these results suggest that the key regulatory step in the mechanism of trophic hormone-stimulated steroid production is the first hydroxylation step of the 3 sequential monooxygenation reactions involved in the conversion of cholesterol to pregnenolone.     

Protein kinase stimulation of a reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.

A solubilized preparation of cytochrome P-450, obtained by treatment of mitochondria from bovine corpora lutea with phospholipase A, contained all of the necessary components for the cholesterol side chain cleavage activity. The solubilized cytochrome -450 preparation could be isolated essentially free of endogenous cholesterol side chain cleavage activity by various fractionation techniques. A cholesterol side chain cleavage enzyme system was reconstituted using the isolated cytochrome P-450 preparation and purified adrenodoxin and adrenodoxin reductase (components of the enzyme system purified from the adrenal cortex). Protein kinase was partially purified from the cytosol fraction of bovine corpora lutea. It was purified 43-fold and the activity was highly dependent on cyclic adenosine 3:5-monophosphate (cyclic AMP). When ATP and this partially purified cyclic AMP-dependent protein kinase were added to the reconstituted cholesterol side chain cleavage enzyme assay in which cytochrome P-450 was limiting, a stimulation (20 to 74%) of the conversion of cholesterol into pregnenolone was observed. This stimulation was statistically significant with p value less than 0.001. The stimulatory effect of the protein kinase appeared to be dependent on ATP and was not mimicked by bovine serum albumin, indicating that the effect was specific for protein kinase. Protein kinase caused a phosphorylation of the cytochrome P-450 preparation when large amounts of this preparation were used in the assay. It is concluded from these results that the direct activation of the cytochrome P-450 component of the cholesterol side chain cleavage by protein kinase may be one of the ways by which cyclic AMP mediates the effect of luteinizine.     

The side-chain cleavage of cholesterol sulfate--II. The effect of phospholipids on the oxidation of the sterol sulfate by inner mitochondrial membranes and by a reconstituted cholesterol desmolase system

This study compares the side-chain cleavage of aqueous suspensions of cholesterol sulfate with the side-chain cleavage of cholesterol sulfate which is incorporated into phospholipid vesicles. Three different cholesterol desmolase systems are examined: the membrane-bound cholesterol side-chain cleavage system present in inner mitochondrial membranes isolated from bovine adrenal mitochondria; a soluble, lipid-depleted, reconstituted side-chain cleavage system prepared from cytochrome P-450scc, adrenodoxin and adrenodoxin reductase; a membrane associated side-chain cleavage system prepared by adding phospholipid vesicles, prepared from adrenal mitochondrial, to the reconstituted system. Soluble cholesterol sulfate, in low concentration, is a good substrate for the lipid-depleted reconstituted side chain cleavage system. However, at concentrations above 2 microM, in the absence of phospholipids, the sterol sulfate appears to bind at a non-productive site on cytochrome P-450scc which leads to substrate inhibition. Phospholipids, while inhibiting the binding of cholesterol sulfate to the cytochrome, also appear to prevent non-productive binding of the sterol sulfate to the cytochrome. Thus the addition of phospholipids to the lipid-depleted enzyme system leads to an activation of side-chain cleavage of high concentrations of the sterol sulfate. Soluble cholesterol sulfate is a good substrate for both the native and reconstituted membrane-bound systems and no substrate inhibition is observed when the membrane bound enzyme systems are employed in the assay of side-chain activity. However, the cleavage of cholesterol sulfate, which is incorporated into phospholipid vesicles, by both membrane bound enzyme systems appears to be competitively inhibited by the phospholipids of the vesicles. The results of this study suggest that the regulation of the side-chain cleavage of cholesterol sulfate may be entirely different than the regulation of the side-chain cleavage of cholesterol, if cholesterol sulfate exists intracellularly as a soluble non-complexed substrate. If, on the other hand, cholesterol sulfate is present in the cell in lipid droplets as a complex with phospholipids, its metabolism may be under the same constraints as the side-chain cleavage of cholesterol.     

Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme

The cholesterol side-chain cleavage enzyme (SCC) catalyzes the initial and rate-limiting step in the synthesis of steroid hormones. The mouse gene encoding SCC was cloned and the nucleotide sequence of its 5'-flanking region determined. This sequence includes an AP-1 motif at -319 and two motifs, AGGTCA at -70 and AGCCTTG at -40, that match elements proposed to be important in the expression of steroid 21-hydroxylase. When transfected into mouse Y1 adrenocortical tumor cells, 1.5 kilobase pairs of 5'-flanking region of the SCC gene directed high levels of expression of a growth hormone reporter gene; treatment of the transfected Y1 cells with 8-bromo-cAMP increased this expression by 5-fold. In contrast, transfected mouse MA-10 Leydig cells showed appreciably lower expression, suggesting that SCC expression in Leydig cells requires additional elements not contained in the 5'-flanking region of the SCC gene used in these experiments. Deletion experiments showed that 424 base pairs of 5'-flanking sequences were sufficient for regulated expression in Y1 cells and mapped two regulatory regions: one from -424 to -327 and a second from -219 to -77. DNase I footprinting and gel mobility shift analyses of these 424 base pairs defined several interactions between nuclear proteins and the SCC promoter, including footprints centered over the AP-1 motif, over a sequence at -120, and over the sequences (-70 and -40) that resemble 21-hydroxylase promoter elements. Finally, site-selected mutagenesis of the potential elements at -40, -70, or -120 decreased SCC promoter activity in transfected Y1 adrenocortical cells, thus establishing their importance in SCC expression.     

Stimulation of rat testis microsomal 17alpha-hydroxyprogesterone side-chain cleavage enzyme by luteinizing hormone added in vitro

The addition of purified luteinizing hormone to isolated rat testis microsomes causes an increase in the activity of the enzyme system that converts 17α-hydroxyprogesterone into C₁₉ steroids in these tissue organelles. The gonadotrophin does not act in this system by altering the Michaelis constants of the enzyme for either steroid or nicotinamide nucleotide, but rather appears specifically to increase the catalytic capability of the protein.     

Clomiphene and tamoxifen inhibit the cholesterol side-chain cleavage enzyme activity in hen granulosa cells

A radiochemical assay was utilized to study the inhibitory effects of clomiphene and tamoxifen on the cholesterol side-chain cleavage enzyme activity in a mitochondrial preparation of granulosa cells isolated from mature ovarian follicles of laying hens. At saturating substrate concentrations, both clomiphene and tamoxifen were able to suppress enzyme activity in a dose-related manner (IC50 1.8 X 10(-5) M). Double reciprocal plots of kinetic data show that the inhibition is mixed, exhibiting competitive kinetics at low concentrations, whereas at high concentrations, the inhibition is of a non-competitive nature. The competitive inhibition constants as determined from Dixon plots are 2 X 10(-5) M for clomiphene and 2.3 X 10(-5) M for tamoxifen. It is concluded that, in granulosa cells, clomiphene and tamoxifen directly inhibit the mitochondrial cholesterol side-chain cleavage activity. This inhibition may represent an important aspect of the mode of action of clomiphene and tamoxifen.     

The side-chain cleavage of cholesterol sulfate--III. The effect of adrenodoxin, membrane phospholipids and Tween 80 on the kinetics of oxidation of the sterol sulfate by a reconstituted cholesterol desmolase system

This paper reports the Km values of a reconstituted cholesterol side-chain cleavage system for cholesterol sulfate, cholesterol, and adrenodoxin, determined under several experimental conditions. The Km values for adrenodoxin change depending on whether cholesterol or its sulfate is used as the substrate. Moreover, the Km values for both of the substrates and for adrenodoxin are greatly modulated by both membrane phospholipids, isolated from adrenal mitochondria, and Tween 80, 0.002%. In the absence of detergents or phospholipids, the enzyme system shows a high affinity for cholesterol sulfate, but is inhibited when high concentrations of the sterol sulfate are added to the incubation mixture. Raising the concentration of adrenodoxin in the assay mixture prevents the substrate inhibition. When cholesterol sulfate is incorporated into micelles containing the phospholipids, the enzyme system does not display substrate inhibition, and the kinetics of cleavage of the sterol sulfate are relatively independent of the concentration of adrenodoxin in the assay mixture. In the absence of phospholipids, the apparent kinetics of cleavage of cholesterol and its sulfate are quite different from each other, but when incorporated into micelles containing phospholipids, the kinetics of cleavage of the two substrates are similar to each other.     

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.     

Stimulation of cholesterol side-chain cleavage by a luteinizing-hormone-releasing hormone (luliberin) agonist (ICI 118630) in rat Leydig cells.

The action of a luliberin (luteinizing-hormone-releasing hormone) agonist (ICI 118630) and lutropin (luteinizing hormone) on the activity of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat Leydig cells has been investigated. This has been carried out by studying the metabolism of exogenous (22R)-22- and 25-hydroxycholesterol to testosterone. It was found that both hydroxycholesterols increased testosterone production to higher levels than achieved by lutropin alone. Addition of luliberin agonist but not lutropin was found to increase further the metabolism of the hydroxycholesterol to testosterone; this occurred in the presence of saturating and subsaturating levels of the hydroxycholesterols. This effect of luliberin agonist was potentiated in the presence of lutropin. The protein synthesis inhibitor, cycloheximide, inhibited the luliberin agonist-induced stimulation of the hydroxycholesterol metabolism. At low calcium levels (1.1 microM), testosterone production was increased by addition of (22R)-22-hydroxycholesterol but the luliberin agonist effect was negated. The calmodulin inhibitor trifluoperazine inhibited (22R)-22-hydroxycholesterol-stimulated steroidogenesis and negated the luliberin agonist effect. These results indicate that luliberin agonist specifically increases the synthesis of the cytochrome P-450 cholesterol side-chain cleavage enzyme in rat testis Leydig cells.     

Formation and functioning of fused cholesterol side-chain cleavage enzymes

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.