Rat P45017αfrom Testis: Characterization of a Full-Length cDNA Encoding a Unique Steroid Hydroxylase Capable of Catalyzing Both Δ4- and Δ5-Steroid-17,20-Lyase Reactions

Hr, Fevold; Mc, Lorence; Jl, McCarthy; Jm, Trant; Kagimoto, Masaaki; Mr, Waterman; Ji, Mason

doi:10.1210/mend-3-6-968

Cited by 157 publications

(55 citation statements)

References 32 publications

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“…RNA was transferred onto nylon membranes (Hybond-N; Amersham International, Aylesbury, Bucks, UK) in 20X sodium citrate/sodium chloride (SSC), by capillary blotting. cDNA probe to rat P-450 cl7a mRNA (full-length cytochrome P-450 C | 7a ; generously donated by Dr J. Ian Mason) (Fevold et al, 1989) was labelled with [ 32 P]dCTP by random priming (Megaprime kit; Amersham). Prehybridization was carried out for 1-2 h at 42°C in 5X saline/sodium phosphate/EDTA (SSPE), 5X Denhardt's solution, 18.5% (v/v) formamide and 0.5% (w/v) sodium dodecyl sulphate (SDS).…”

Section: Northern Analysis Of Cytochrome P-450 Cl7a Mrnamentioning

confidence: 99%

Effect of luteinizing hormone on follicle stimulating hormone-activated paracrine signalling in rat ovary

Smyth¹,

Miró²,

Howles³

et al. 1995

Human Reproduction

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'Pure' follicle stimulating hormone (FSH) and luteinizing hormone (LH) are expected shortly to become available for pharmaceutical use in the clinical setting. To test the contribution of LH to optimal ovarian responsiveness to FSH, 21-day-old hypophysectomized, immature, female rats received four s.c. injections of recombinant human LH (rhLH; total dose 1-10 IU) and/or rhFSH (total dose 30-72 IU) given at 12-hourly intervals. At 48 h after the first injection, ovaries were removed, weighed and used to isolate granulosa and thecal/interstitial cells for assessment of basal and gonadotrophin-responsive steroidogenesis in vitro, or homogenized to extract total RNA for Northern analysis of 17-hydroxylase/Ci7_ 2 o-lyase (cytochrome P-450 C | 7a ) mRNA. Serum oestradiol and uterine weight were measured as indices of ovarian oestrogen production; androstenedione was measured to reflect ovarian androgen production. Consistent with the two-cell, two-gonadotrophin model of oestrogen synthesis, increased ovarian oestrogen secretion only occurred if both rhFSH and rhLH were given simultaneously. Treatment with rhFSH alone stimulated ovarian weight gain and granulosa cell aromatase activity without oestrogen secretion, whereas rhLH alone stimulated thecal androgen synthesis and androgen secretion. When the total rhLH dose was fixed at 1 IU, giving rise to an unmeasurably low serum concentration of rhLH, additional treatment with rhFSH (30-72 IU) dose-dependently stimulated serum androgen concentrations as well as oestrogen concentrations. The -2.0 kbsized P-450 C | 7a mRNA transcript was undetectable in the ovaries of untreated control animals but was abundant in the ovaries of positive controls treated with 15 IU of pregnant mare serum gonadotrophin. Treatment with 1 IU of rhLH alone barely induced a P-450 C | 7a mRNA signal and treatment with 30 IU of rhFSH alone was completely ineffective. However, combined treatment with 1 IU of rhLH and 30 IU of rhFSH markedly enhanced the P-450 d7a mRNA signal to a level approaching the positivecontrol. Since P-450 C | 7ct mRNA is expressed exclusively in thecal cells, which do not possess FSH receptors, we conclude that (i) rhFSH upregulates thecal P-450 d7a mRNA and hence follicular androgen synthesis via granulosa-on-

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Section: Northern Analysis Of Cytochrome P-450 Cl7a Mrnamentioning

confidence: 99%

Effect of luteinizing hormone on follicle stimulating hormone-activated paracrine signalling in rat ovary

Smyth¹,

Miró²,

Howles³

et al. 1995

Human Reproduction

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“…Specifically addressed in this report is a particularly intriguing aspect of CYP17A1 structure, wherein the choice of substrate controls hydrogen bonding that defines the catalytic channel for product formation. [4][5][6]15] Herein rR spectroscopy convincingly demonstrates that the single difference at the 3 position of OH-PREG and OH-PROG leads to unequivocal changes in active site H-bonding interactions with the key Fe-O-O fragment of enzymatic intermediates, leading to alterations in electronic structure that then control substrate processing. Figure 2A are the rR spectra obtained for the 16 (Figure 2A).…”

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confidence: 89%

“…In vivo, the predominant pathway forming androgens proceeds through the conversion of hydroxypregnenolone to dehydroepiandrosterone. [4][5][6] We report newly acquired resonance Raman (rR) spectra of monomeric CYP17A1 self-assembled in Nanodiscs, which reveal a distinct difference in hydrogen bonding to the ferrous dioxygen intermediate. With 17-hydroxyprogesterone, the oxygen vibrational modes indicate hydrogen bonding to the distal oxygen of the Fe-O-O fragment, whereas with 17-hydroxypregnenolone hydrogen bonding is to the proximal oxygen.…”

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confidence: 99%

Differential Hydrogen Bonding in Human CYP17 Dictates Hydroxylation versus Lyase Chemistry

et al. 2013

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Raman spectra of oxygenated intermediates of Nanodisc incorporated human CYP17 in the presence of natural substrates directly confirm that substrate structure effectively alters H-bonding interactions with the critical Fe-O-O fragment so as to dictate its predisposition for one of two alternative reaction pathways, providing a realistic structural explanation for substrate control of CYP17 reactivity that has profound physiological implications. Keywordsbiophysics; heme proteins; oxidoreductases; Raman spectroscopy; lyase reaction CYP17A1 is a member of the cytochrome P450 superfamily that occupies a central role in the biosynthesis of steroid hormones. [1,2] This enzyme catalyzes both the hydroxylation of its primary substrates pregnenolone and progesterone as well as the subsequent 17,20 carbon-carbon lyase chemistry which is the first committed step in the biosynthesis of androgens. [1][2][3] There is significant debate as to the chemical mechanisms of this lyase activity, with human CYP17 exhibiting a significant preference for 17-hydroxy pregnenolone over 17-hydroxy progesterone. In vivo, the predominant pathway forming androgens proceeds through the conversion of hydroxypregnenolone to dehydroepiandrosterone. [4][5][6] We report newly acquired resonance Raman (rR) spectra of monomeric CYP17A1 self-assembled in Nanodiscs, which reveal a distinct difference in hydrogen bonding to the ferrous dioxygen intermediate. With 17-hydroxyprogesterone, the oxygen vibrational modes indicate hydrogen bonding to the distal oxygen of the Fe-O-O fragment, whereas with 17-hydroxypregnenolone hydrogen bonding is to the proximal oxygen. To the extent that such interactions persist in the subsequent peroxo-intermediate, the latter interaction is expected to inhibit O-O bond cleavage relative to the former,

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“…Cortisol production in the ZF is associated with abundant expression of both P450c17 and HSD3B2 (Ishimura & Fujita 1997), in addition to P450c21 and P450c11. Under basal conditions at least, studies of the expressed cDNA have shown that the human P450c17 prefers P5 as a substrate over progesterone (P4) and that the hydroxylase activity is both faster and has higher affinity for binding of substrate than the 17,20-lyase reaction (Voutilainen & Miller 1986, Fevold et al 1989, Brock & Waterman 1999, Flück et al 2003. Since only 17-hydroxylation activity is required of P450c17 for cortisol biosynthesis, the additional presence of abundant HSD3B2 in the ZF cannot prevent this initial hydroxylase reaction, but can effectively compete for 17OHP5 and convert it to 17OHP4.…”

Section: Adrenal Zonation and Zonal Functionmentioning

confidence: 99%

Plasticity of the zona reticularis in the adult marmoset adrenal cortex: voyages of discovery in the New World

Pattison¹,

Abbott²,

Saltzman³

et al. 2009

Journal of Endocrinology

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Adrenarche in humans occurs at the age of 5-7 years, yet the process by which dehydroepiandrosterone (DHEA) biosynthesis in the adrenal zona reticularis (ZR) increases so dramatically remains as a matter of debate. One suggestion is that increased DHEA production by P450c17 (CYP17A1 as listed in HUGO Database) in the ZR results from a coincident fall in the expression of HSD3B, which would otherwise compete for pregnenolone substrate. Nonetheless, studies of human and rhesus adrenal show that cytochrome b5 (CYTB5) expression increases in the ZR with DHEA biosynthesis, and cloned human and rhesus P450c17 show selective increases in 17,20-lyase activity in the presence of CYTB5. The marmoset, a New World primate, expresses a fetal zone during development which regresses after birth. Adult males, however, do not develop an obvious functional ZR, while females develop a ZR in a manner that depends on their social/gonadal status. In all social and physiologic states, changes in marmoset ZR function relate directly to changes in the expression of CYTB5. Recent cloning and expression of marmoset P450c17 also show that while amino acid sequence homology is in the order of w85% of that found in human and rhesus sequences, and basal lyase activity is low compared with rhesus, all previously described amino acids critical to human 17,20-lyase activity are completely conserved. Furthermore, the 17,20-lyase activity of the marmoset P450c17 clone is dramatically increased by addition of CYTB5. We propose that these combined data from the marmoset model provide further compelling evidence that the control of ZR CYTB5 expression is a key determinant of ZR function.

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Rat P450_17αfrom Testis: Characterization of a Full-Length cDNA Encoding a Unique Steroid Hydroxylase Capable of Catalyzing Both Δ⁴- and Δ⁵-Steroid-17,20-Lyase Reactions

Cited by 157 publications

References 32 publications

Effect of luteinizing hormone on follicle stimulating hormone-activated paracrine signalling in rat ovary

Effect of luteinizing hormone on follicle stimulating hormone-activated paracrine signalling in rat ovary

Differential Hydrogen Bonding in Human CYP17 Dictates Hydroxylation versus Lyase Chemistry

Plasticity of the zona reticularis in the adult marmoset adrenal cortex: voyages of discovery in the New World

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