Molecular Cloning and Expression of Guinea Pig Cytochrome P450C17 cDNA (Steroid 17α-Hydroxylase/17,20 Lyase): Tissue Distribution, Regulation, and Substrate Specificity of the Expressed Enzyme

Abstract

In mammalian and fish species, P450c17 mediates both 17 alpha-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones. Previous results have shown that among the adrenal steroid hydroxylase enzymes involved in adrenal C19 steroid and glucocorticoid synthesis, regulation of cytochrome P450c17 is of primary importance because it is localized at the key branch between glucocorticoid and C19 steroid synthesis. A cDNA library from guinea pig adrenal was constructed, and the complete 17 alpha-hydroxylase cytochrome P450 cDNA was isolated. The guinea pig P450c17 cDNA includes the full-length coding region (1,524 nucleotide), the complete 3' untranslated region (169 nucleotide), and 39 bases of the 5' untranslated region. Our clone shares most of the features of the other P450c17 cDNAs; however, in addition, we identified a novel conserved region of 18 amino acids located in exon I between residues 80 and 97. This region presents the highest percentage of identity among the other P450c17 enzymes and is positioned one helixturn upstream of the important Ser106 on the corresponding human form. On Northern blot, the cDNA hybridizes with a major 1.8-kb mRNA and with two other related P450c17 mRNA of about 3 and 4 kb. P450c17 mRNA is equally distributed in male and female gonads and adrenals. Characterization of the enzymatic activity shows that 17 alpha-hydroxylase and 17,20-lyase are carried by a single protein, but in homogenates 17,20-lyase activity is barely detectable. Moreover, we demonstrate in vitro and in vivo that the guinea pig enzyme preferentially has very high levels of 17 alpha-hydroxylase and 17,20-lyase activities only toward delta 4 steroids. Second-messenger cyclic adenosine monophosphate and adrenocorticotropin specifically increased the abundance of P450c17 mRNA levels in guinea pig adrenal cells.

Vasotocin and Mesotocin Stimulate the Biosynthesis of Neurosteroids in the Frog Brain

“…The animals were housed in a temperature-controlled room (8 Ϯ 0.5°C) under running water, on a 12 h light/dark schedule (lights on from 6:00 A.M. to 6:00 P.M.), for at least 1 week before use. To limit possible variations of neurosteroid biosynthesis attributable to circadian rhythms (Akwa et al, 1991) (Tremblay et al, 1994) as well as a monoclonal antibody against VP (Robert et al, 1985) were used as primary antibodies for immunohistochemistry. Alexa 594-conjugated goat anti-rabbit ␥-globulins (GARs/Alexa 594) and Alexa 488-conjugated goat anti-mouse ␥-globulins (GAMs/Alexa 488), both from Invitrogen (Carlsbad, CA), were used as secondary antibodies.…”

Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa

“…Rabbit antisera were raised against purified bovine testicular CYP17 [31], and against human placental 3␤HSD (type I) [32] and 17␤HSD (typeI) [33]. These antisera were used previously to localize CYP17-, 3␤HSD-and 17␤HSD-positive cells in the brain of the frog Rana esculenta [34,35] and Rana ridibunda [36].…”

Immunoelectron microscopic localization of three key steroidogenic enzymes (cytochrome P450(scc), 3 beta-hydroxysteroid dehydrogenase and cytochrome P450(c17)) in rat adrenal cortex and gonads

“…They were immunostained using protein A-gold complex (10 nm; British Biocell Int., Cardiff, UK), as described (Roth et al 1978). The antisera to human P450 scc (supplied by Dr Miller;Black et al 1993), human type 1 3 -HSD (Luu-The et al 1989, 1990, Pelletier et al 1992 and human P450 c17 (Tremblay et al 1994) were all used at dilutions ranging from 1:500 to 1:1000. Control experiments were performed by substituting non-immunized rabbit serum (1:500) or the antiserum (1:500) absorbed with an excess of their respective antigen (10 −6 M).…”