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PubMedDate not supplied

Partial 17, 20-desmolase and 17 alpha-hydroxylase deficiencies in a 16-year-old boy.

Thirteen plasma steroids as well as ACTH, LH and FSH were measured by specific RIAs under basal and dynamic conditions in a 16-year-old boy (normal external genitalia, 46, XY karyotype) who presented slowness and unachievement of pubertal development. On the delta 4-pathway: basal levels of testosterone and dihydrotestosterone were low- with a normal ratio-, delta 4-androstenedione and 11 beta-hydroxyandrostenedione were in the low normal range. Meanwhile, 17 alpha-hydroxyprogesterone and progesterone levels were markedly elevated. On the delta 5-pathway: dehydroepiandrosterone was extremely low while 17 alpha-hydroxypregnenolone and pregnenolone were almost normal; dehydroepiandrosterone sulfate was subnormal while pregnenolone sulfate was normal. Cortisol, aldosterone were normal while ACTH was moderately increased. Basal and responsive levels of LH and FSH were markedly increased. ACTH stimulation induced a subnormal rise of cortisol and 11 beta-hydroxyandrostenedione, a low or absent rise of dehydroepiandrosterone, 17 alpha-hydroxypregnenolone, androstenedione and 17 alpha-hydroxyprogesterone contrasting with a marked rise of pregnenolone and progesterone. After hCG stimulation, responses were low for testosterone, extremely high for 17 alpha-hydroxyprogesterone with a normalisation of the 17 alpha-hydroxyprogesterone/progesterone ratio. Fluoxymesterone dramatically reduced the pathologically high basal levels of progesterone and 17 alpha hydroxyprogesterone. Dexamethasone induced only a minute decrease in the delta 4-progestagens, a marked decrease in pregnenolone, with a more than 80% reduction of 17 alpha- hydroxypregnenolone, dehydroepiandrosterone, dehydroepiandrosterone sulfate and androstenedione. These data suggest a defect involving the cytochrome P450 common to both 17 alpha-hydroxylase and 17, 20-desmolase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1985

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.

Adrenal Glands↗
PubMed1995

The rat 17 alpha-hydroxylase-17,20-desmolase (CYP17) active site: computerized homology modeling and site directed mutagenesis.

A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP17) steroid binding domain was derived from the alpha/beta F supersecondary structural element of the 3 alpha/20 beta hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding. These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence. The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431. Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element. These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote alpha/beta F supersecondary element.

Aldehyde-Lyases↗
PubMed1999

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.

Adult↗
PubMed1987

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.

Base Sequence↗
PubMed1991

Structures of regulatory regions in the human cytochrome P-450scc (desmolase) gene.

The mechanism of regulatory expression of human cytochrome P-450scc gene by cAMP was investigated in a transient expression system using Y-1 cells (mouse adrenal tumor cell line) and a chimeric DNA composed of the structural gene for bacterial chloramphenicol acetyltransferase and the 5' flanking upstream sequence of the cytochrome P-450scc (cholesterol desmolase) gene which was revealed to contain a DNA element(s) responsive to cAMP [Inoue, H. et al. (1988) Eur. J. Biochem. 171, 435-440]. Introduction of deletions and point mutations in the upstream regulatory sequence demonstrated that three regions were mainly required for response to cAMP. These regions contained a short similar sequence. All of them have a 5-bp motif GTCAT (or ATGAC) in common, and have at least two motifs which conserve four out of five base pairs of the consensus sequence of the cAMP-responsive element (CRE), CGTCA (or TGACG). They are all apparently necessary for regulation by cAMP. Gel mobility shift assays suggested that a binding factor(s) to these regions was present in the nuclear extracts of Y-1 cells and adrenal cortex tissues and appeared to be different from the somatostatin CRE-binding protein. Deletion analysis also suggested that the region around -44 was essential to the basal transcriptional activity. This region shows some similarity to the CTF NF-1 binding site [Johnson and McKnight (1989) Annu. Rev. Biochem. 58, 799-839].

Animals↗
PubMed1986

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.

20-Hydroxysteroid Dehydrogenases↗
PubMed1982

Dexamethasone-suppressible hypercorticosteronism in two 46,XX subjects with ambiguous genitalia and ovarian cysts. Partial defect of 17 alpha-hydroxylase or 17-20-desmolase.

The paradoxical association of female pseudohermaphroditism and androgen deficiency was observed in two 46,XX subjects with high corticosterone plasma levels. Subject 1 has been declared a boy due to clitoris enlargement; she had no vagina and uterus. Subject 2 had ambiguous external genitalia. In both, at age 27 and 17 years, fusion of outer labia, impuberism, ovarian cysts, and histologically normal ovarian tissue were observed. Blood pressure was normal. Basal cortisol levels were normal but unresponsive to ACTH. Progesterone levels were 40 and 62 ng/ml and rose after ACTH (50 and 79 ng/ml). 17-hydroxyprogesterone levels were 25 and 21 ng/ml and did not rise after ACTH. Corticosterone levels were 70 and 92 ng/ml and rose after ACTH (110 and 180 ng/ml). All three steroids were suppressed by dexamethasone. Androgen and estrogen levels were at or below the lower limit for normal women. The sex steroid levels obtained by radioimmunoassay in plasma and a follicular cyst fluid were confirmed by isotope dilution-mass spectrometry. We suggest that the sexual ambiguousness resulted from an excessive production of gestagenic steroids during fetal life, and that the enzyme defect is either a partial 17 alpha-hydroxylase defect combined with a peripheral production of 17-hydroxyprogesterone, or else a partial 17-20-desmolase defect with a secondary 21-hydroxylase defect limited to the cortisol pathway.

Adolescent↗
PubMed1986

Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes.

Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.

Adrenal Hyperplasia, Congenital↗
PubMed1980

Familial male pseudohermaphroditism due to 17-20-desmolase deficiency. I. In vivo endocrine studies.

In two siblings with male pseudohermaphroditism (ambiguous external genitalia, XY karyotype) and apparently normal glucocorticoid function, plasma concentrations of 10 progestagens or androgens measured by specific RIAs were found to be abnormal under either basal or dynamic conditions. Basal levels of delta 4-androstenedione, dehydroepiandrosterone, and dehydroepiandrosterone sulfate were subnormal and failed to rise after ACTH stimulation both before and after castration. Meanwhile, levels of pregnenolone, pregnenolone sulfate, 17 alpha-hydroxyprogesterone, and 17 alpha-hydroxypregnenolone were extremely high under basal conditions and rose further after ACTH. All of the progestagens and cortisol were suppressed by dexamethasone. After hCG stimulation, either before treatment or during dexamethasone therapy, the rise in testosterone was less than 100 ng/dl, while the progestagens showed an abnormally high rise. The latter were markedly reduced after castration. These findings are consistent with steroid 17--20-desmolase deficiency in both the testes and adrenal glands. In the third brother, who had only slight abnormalities of his genitalia, a mild form of the same defect was suspected. Low androgens, high 17 alpha-hydroxypregnenolone, and 17 alpha-hydroxyprogesterone levels were found in the amniotic fluid and umbilical cord and peripheral blood at birth. The parents, who were not consanguine, had normal baseline levels of all hormones. The familial occurrence of the disease is suggestive of autosomal recessive inheritance.

17-alpha-Hydroxypregnenolone↗
PubMed1983

Male pseudohermaphroditism due to 17,20-desmolase deficiency.

In a 5-yr-old 46,XY male pseudohermaphrodite with microphallus, perineal hypospadias, chordee and cryptorchidism, serum C19 steroid levels were abnormally low in the basal state and after adrenal and testicular stimulation. Serum C21 steroid levels were elevated in the basal state and increased further after adrenal, but not after gonadal, stimulation. Urinary excretion of pregnanetriolone, a metabolite of 17-hydroxypregnenolone and 17-hydroxyprogesterone not normally present in the urine, was increased in the basal and stimulated states. Cortisol production was normal, and all steroid hormone levels were suppressed by dexamethasone. Testicular biopsy was consistent with prepubertal cryptorchid testes. Incubation of testicular tissue with labeled 17-hydroxyprogesterone revealed failure of conversion of precursor to androstenedione and testosterone. A significant increase in phallic length occurred after treatment with exogenous androgen. These findings are consistent with 17,20-desmolase deficiency in both gonads and adrenal glands.

Adolescent↗
PubMed1979

In vitro C-20, 22 desmolase regulation of pregnenolone supply in first trimester human placenta.

Homogenates of first trimester human placentae were incubated with [26-14C]chloresterol. Using reverse-isotope dilution analysis [14C]isocaproic acid was isolated and characterized. A progressive increase in [14C]isocaproic acid formation with increasing gestation suggestive of a regulatory mechanism at the C-20, 22 desmolase site in the supply of placental pregnenolone was noted.

Caproates↗
PubMed1986

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.

Adrenal Cortex↗
PubMed1982

Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11 beta-hydroxylase) from bovine adrenal cortex.

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11 beta-hydroxylation (11 beta)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11 beta was able to incorporate phosphate from ATP in the presence of C (Km = 7.5 microM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11 beta (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11 beta-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11 beta phosphorylation strikingly increases the P-450 11 beta-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11 beta-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.

Adrenal Cortex↗