The concentration of adrenodoxin reductase limits cytochrome P450scc activity in the human placenta

Abstract

We have previously reported that cytochrome P450scc activity in the human placenta is limited by the supply of electrons to the P450scc [Tuckey, R. C., Woods, S. T. & Tajbakhsh, M. (1997) Eur. J. Biochem. 244, 835±839]. The aim of the present study was to determine whether it is adrenodoxin reductase, adrenodoxin or both which limits cytochrome P450scc activity and hence progesterone synthesis in the placenta. We found that the concentrations of adrenodoxin reductase and adrenodoxin in placental mitochondria were both considerably lower than the concentrations of these proteins in the bovine adrenal cortex. When P450scc activity assays were carried out at high mitochondrial protein concentrations, we found that the addition of exogenous adrenodoxin reductase to sonicated mitochondria rescued pregnenolone synthesis to a level above that for intact mitochondria, showing that adrenodoxin is near-saturating in vivo. In contrast, pregnenolone synthesis by sonicated mitochondria was almost zero even after the addition of human adrenodoxin. This shows that the concentration of endogenous adrenodoxin reductase was insufficient to support appreciable rates of pregnenolone synthesis, even when concentrated mitochondrial samples were used. Comparative studies with human and bovine adrenodoxin reductase have revealed that a twofold higher concentration of human adrenodoxin reductase is required for maximal P450scc activity in the presence of saturating human adrenodoxin. Thus, not only is the adrenodoxin concentration low in placental mitochondria, but the amount required for maximal P450scc activity is higher than that for the bovine reductase. Overall, the data indicate that the adrenodoxin reductase concentration limits the activity of P450scc in placental mitochondria and hence determines the rate of progesterone synthesis.

A novel pathway for sequential transformation of 7‐dehydrocholesterol and expression of the P450scc system in mammalian skin

“…The calculated rates of conversion of [4][5][6][7][8][9][10][11][12][13][14] C]cholesterol into pregnenolone and progesterone in cutaneous mitochondria were 0.14% and 0.04%, respectively, 1% of the conversion reported for placental mitochondria [39]. Pregnenolone was also detected by RIA in the culture medium of skin cells incubated for 18 h with 25 lM 22R-hydroxycholesterol (not shown).…”

“…Lastly, when mitochondria from skin cells (immortalized and malignant keratinocytes) were incubated with [4][5][6][7][8][9][10][11][12][13][14] C]cholesterol, it resulted in the production of steroids that migrated at the same rate as the pregnenolone and progesterone standards (not shown). The calculated rates of conversion of [4][5][6][7][8][9][10][11][12][13][14] C]cholesterol into pregnenolone and progesterone in cutaneous mitochondria were 0.14% and 0.04%, respectively, 1% of the conversion reported for placental mitochondria [39].…”

“…8. Transformation of 7-DHC (1) to 7-DHP (2), followed by a proposed sequence for the enzymatic transformation of 7-DHP to its hydroxy derivatives (3)(4)(5)(6)(7)(8)(9)(10), and/or to secosteroids (11)(12)(13)(14)(15)(16)(17)(18)(19) generated by the action of UVB radiation. 3, 3b,11a-or b-Dihydroxypregna-5,7-dien-20-one; 4, 3b,17b-dihydroxypregna-5,7-dien-20-one; 5, 3b,21-dihydroxypregna-5,7-dien-20-one; 6, 3b,17b,21-trihydroxypregna-5,7-dien-20-one; 7, 3b,11a-or 11b,21-trihydroxypregna-5,7-diene-20-one; 8, 3b,11a-or 11b,17-trihydroxypregna-5,7-diene-20-one; 9, 3b,11a-or b,17b,21-tetrahydroxypregna-5,7-dien-20-one; 10, 3b,20a-or b-dihydroxypregna-5,7-diene; 11, 5Z,7E-3b-hydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 12, 5Z,7E-3b, 11a-or b-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 13, 5Z,7E-3,17b-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 14, 5Z,7E-3,b,21-dihydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 15, 5Z,7E-3b,17b,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 16, 5Z,7E-3b,11a-or 11b,21-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 17, 5Z,7E-3b,11a-or 11b,17-trihydroxy-9,10-secopregna-5,7,10(19)trien-20-one; 18, 5Z,7E-3b,11a-or b,17b,21-tetrahydroxy-9,10-secopregna-5,7,10(19)-trien-20-one; 19, 5Z,7E-3b,11a-or b-dihydroxy-9,10-secopregna-5,7,10(19)triene.…”

“…The reaction takes place at a single active site on the cytochrome to produce pregnenolone [8]. Electrons for the hydroxylations are provided by NADPH through the electron transfer proteins adrenodoxin reductase and adrenodoxin [8,9]. This biochemical pathway may be operative in the skin, as it expresses the related CYP11A1, CYP17, CYP21A2 and MC2-R genes [10].…”

Hydroxylation of CYP11A1-Derived Products of Vitamin D3 Metabolism by Human and Mouse CYP27B1

“…Mouse and human adrenodoxin and human adrenodoxin reductase were expressed in E. coli and purified as before (Woods et al, 1998;Tuckey and Sadleir, 1999;Tang et al, 2010bTang et al, , 2012. Mouse and human CYP27B1 were coexpressed in E. coli with the chaperonins, GroEL/ES, to facilitate correct protein folding, as described before (Tang et al, 2010b.…”