Corticotropin-induced changes in a soluble desmolase preparation

Following simple homogenization, substantial desmolase activity is recovered in rat adrenal 105 000 × g supernatant. The desmolase complex sediments at 3–4 S on sucrose gradients, is found in the clear cytosol, requires NADPH, is derived from mitochondria and is inhibited by aminoglutethimide and pregnenolone. The lipid fraction contains little or no desmolase activity but greatly enhances pregnenolone synthesis in soluble desmolase preparations, presumably by supplying free cholesterol substrate. Prior adrenocorticotropin (ACTH) administration enhances pregnenolone synthesis in the 105 000 × g supernatant, and cycloheximide, an inhibitor of adrenal protein synthesis, does not block this effect of ACTH (but rather potentiates it). The ACTH effect may be largely explained by an increase in free cholesterol, which enhances the activity of both the lipid fraction and clear cytosol, since: free cholesterol levels are increased by ACTH, particularly with cycloheximide pretreatment; type I and inverted type I difference spectrum changes, indicating greater cholesterol availability for binding to cytochrome P-450, are enhanced by ACTH with or without cycloheximide treatment; cholesterol-rich lipid fraction enhances such spectral changes and obliterates the differences in spectral and pregnenolone-synthesizing activities betwen control and ACTH-stimulated soluble desmolase preparations; and desmolase stimulatory properties of clear cytosol co-chromatographs with [¹⁴C]cholesterol. Since cycloheximide blocks ACTH-induced effects in intact mitochondria but not in the soluble desmolase preparation, it is postulated that the labile protein required during ACTH action functions to overcome a ?restraining influence’ which is present in intact mitochondria but not in the soluble desmolase system. The ‘restraining influence’ may be due to limited cholesterol-desmolase interaction.     

Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris

The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase.     

Steroid desmolase synthesis by Eubacterium desmolans and Clostridium cadavaris.

The synthesis of a steroid desmolase was demonstrated in two obligate anaerobes: a new bacterial species, Eubacterium desmolans, isolated from cat fecal flora, and Clostridium cadavaris, recovered from sewage of New York City. The enzyme cleaves the C-17-C-20 bond of corticoids possessing hydroxyl functions at C-17 and C-21. The conversion is quantitative, provided the substrate concentration is less than 100 micrograms/ml and the organisms are in the log phase. The velocity of transformation parallels the bacterial growth curve and in the log phase is higher for E. desmolans than for C. cadavaris. In addition, both organisms synthesize a 20 beta-hydroxysteroid dehydrogenase.     

Gene structure of human cytochrome P-450(SCC), cholesterol desmolase

Four independent clones containing a part of the P-450(SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450(SCC) cDNA as a probe. These clones covered the entire P-450(SCC) gene except for a part of the 1st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72% homology with that of bovine P-450(SCC). A CATT sequence and a TATAAT sequence, which are possibly a "CAT" box, and a "TATA" box, respectively, are present 129 and 91 bp upstream from the initiation codon. An unusual exon/intron junctional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450(SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450(SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450(SCC) precursor. The mature form of human P-450(SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5th ligand to the heme.     

Corticotropin induced synthesis of the cholesterol desmolase enzymatic complex in cultured adrenal cortex cells.

Basing on the use of highly specific antibodies it has been proved that ACTH induces synthesis of cytochrome P-450 and adrenodoxin in cultured adrenal cortex cells.     

Expression of C-20, 22 desmolase activity by the human fallopian tube in vitro: evidence for steroidogenesis.

With a view to establishing whether cells of the human Fallopian tubes possess the cholesterol side-chain cleavage activity, homogenates of the tubes, obtained from 6 women (39-45 years) following abdominal hysterectomy for benign conditions, were incubated with (7n-3H)-cholesterol as substrate. Controls (n=6, age 40-44 years) were homogenates heated in a boiling water bath for 10 min. Using the reverse-isotope dilution technique, (3H)-pregnenolone was isolated and characterized. No such metabolite was evident in control incubations of heat-denatured enzymes. The extent of enzymic conversion varied from 1.9 x 10(-3) to 1.3 x 10(-2)%. The results reveal for the first time the existence of an active cholesterol-specific C-20, 22 desmolase system in the viable tissues. It is suggested that there exists a potential for substantial pregnenolone synthesis in vivo. This rate-limiting steroid biosynthetic conversion provides a new dimension to the functional capacity of the Fallopian tubes in the synthesis of steroids, which may be necessary for modulating ciliary beat frequency and in maintenance of hormonal milieu essential for embryogenesis.     

Mode of action of steroid desmolase and reductases synthesized by Clostridium "scindens" (formerly Clostridium strain 19)

A recently isolated hitherto unknown Clostridium from human feces, designated Clostridium "scindens" (formerly strain 19), synthesizes at least two enzymes active on the side-chain of the steroid molecule and two enzymes active on the hydroxyl groups of the 7-position of bile acids. Steroid desmolase, responsible for side-chain cleavage of corticoids, and 20 alpha-hydroxysteroid dehydrogenase have not been detected in any other bacterial species of the resident colonic flora. Steroid desmolase is Eh-dependent (optimum ca. -130 mV), requires a hydroxy group at C-17, and preferably an alpha-ketol group in the side-chain; an alpha-hydroxy group at C-20 reduces and a beta-hydroxy group at C-20 prevents side-chain cleavage. With suitable substrates, the yield of C-19 steroids is proportional to the bacterial multiplication rate. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSDH) is also Eh-dependent (optimum ca. -300 mV) and reduces the C-20 keto function to an alpha-hydroxy group, regardless of the presence or absence of a hydroxy group at C-17. 7 alpha-Dehydroxylase metabolizes cholic and chenodeoxycholic acid, while 7 beta-hydroxysteroid dehydrogenase acts upon ursodeoxycholic acid. The latter two enzymes are not specific for C. scindens.     

On the requirement for protein synthesis during corticotropin-induced stimulation of cholesterol side chain cleavage in rat adrenal mitochondrial and solubilized desmolase preparations.

Following simple homogenization, significant amounts of mitochondrial-derived, cholesterol side chain cleaving enzyme (desmolase) activity are recovered in rat adrenal 105 000 X g-supernatant fraction. Corticotropin administration enhances soluble desmolase activity, and cycloheximide potentiates this effect. The lipid droplet fraction which has no desmolase activity markedly enhances pregnenolone synthesis in the soluble desmolase preparations, presumably by supplying free cholesterol substrate. Corticotropin particularly with cycloheximide pretreatment, enhances lipid fraction activity. Thus increased cholesterol availability may largely explain the corticotropin effect on the soluble desmolase system. Since protein synthesis is required for corticotropin activity in intact mitochondria, but not in calcium-swollen mitochondria or the soluble enzyme system, the labile protein apparently required during corticotropin action may function to overcome a "barrier" which exists only in the intact mitochondria and restrains cholesterol side chain cleavage.     

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.     

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.