Electron paramagnetic resonance studies of cytochrome P-450 and adrenal ferredoxin in single whole rat adrenal glands. Effect of corticotropin

Williams-Smith, D. L.; Simpson, Evan R.; Barlow, Susan; Morrison, P. J.

doi:10.1016/0005-2728(76)90008-6

Cited by 39 publications

(8 citation statements)

References 19 publications

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“…In the rat adrenal cortex the adrenodoxin pool in the mitochondria appears to be almost fully reduced during active steroidogenesis, indicating that reduction of Adx by AR is not rate limiting [30]. If AR is limiting for P450scc activity in the human placenta, as indicated by the present study, then the Adx pool would be expected to be largely oxidized during P450scc catalysis.…”

Section: Discussionmentioning

confidence: 67%

“…If AR is limiting for P450scc activity in the human placenta, as indicated by the present study, then the Adx pool would be expected to be largely oxidized during P450scc catalysis. The study on the extent of Adx reduction in the rat adrenal involved EPR of whole adrenals taken from rats subjected to different treatment regimes [30]. Such a study is not possible with the human placenta.…”

Section: Discussionmentioning

confidence: 99%

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

Tuckey

Sadleir

1999

European Journal of Biochemistry

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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.

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

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

Tuckey

Sadleir

1999

European Journal of Biochemistry

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“…These workers reported that under nonturnover conditions, oxidized Adx binds to oxidized P450scc with twice the affinity of reduced Adx. Adx has been reported to be fully reduced in the adrenal cortex under steady state conditions so competitive inhibition by oxidized Adx is unlikely to play a regulatory role in this tissue [22]. Hanukoglu and Jefcoate [6], using bovine proteins in a reconstituted system employing Tween 20, have reported a direct correlation between the proportion of total Adx that is reduced and P450scc activity.…”

Section: Discussionmentioning

confidence: 99%

Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta

Tuckey¹,

McKinley

Headlam³

2001

European Journal of Biochemistry

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The conversion of cholesterol to pregnenolone by cytochrome P450scc is the rate‐determining step in placental progesterone synthesis. The limiting component for placental cytochrome P450scc activity is the concentration of adrenodoxin reductase in the mitochondria, where it permits cytochrome P450scc to work at only 16% of maximum velocity. Adrenodoxin reductase serves to reduce adrenodoxin as part of the electron transfer from NADPH to cytochrome P450scc. We therefore measured the proportion of adrenodoxin in the reduced form in intact mitochondria from the human placenta during active pregnenolone synthesis, using EPR. We found that the adrenodoxin pool was only 30% reduced, indicating that the adrenodoxin reductase concentration was insufficient to maintain the adrenodoxin in the fully reduced state. As both oxidized and reduced adrenodoxin can bind to cytochrome P450scc we tested the ability of oxidized adrenodoxin to act as a competitive inhibitor of pregnenolone synthesis. This was done in a fully reconstituted system comprising 0.3% Tween 20 and purified proteins, and in a partially reconstituted system comprising submitochondrial particles, purified adrenodoxin and adrenodoxin reductase. We found that oxidized adrenodoxin is an effective competitive inhibitor of placental cytochrome P450scc with a Ki value half that of the Km for reduced adrenodoxin. We conclude that the limiting concentration of adrenodoxin reductase present in placental mitochondria has a two‐fold effect on cytochrome P450scc activity. It limits the amount of reduced adrenodoxin that is available to donate electrons to cytochrome P450scc and the oxidized adrenodoxin that remains, competitively inhibits the cytochrome.

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“…The rate-limiting enzymatic reaction in steroid biosynthesis (i.e., the conversion of free cholesterol to pregnenolone) is catalyzed by the cytochrome P450 side chain cleavage enzyme (CYP11A1; herein called P450 scc ) [1][2][3]. However, the physiologically relevant rate-limiting step is the transfer of free cholesterol from the outer mitochondrial membrane to the inner membrane where the P450 scc resides [4].…”

Section: Introductionmentioning

confidence: 99%

Involvement of Protein Kinase C and Cyclic Adenosine 3′,5′-Monophosphate-Dependent Kinase in Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis in Leydig Cells1

King

Khan

et al. 2005

Biology of Reproduction

102

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This study investigated the roles of the protein kinase C (PKC) and protein kinase A (PKA) pathways in regulating constitutive steroidogenesis and steroidogenic acute regulatory (STAR; herein designated by its common name, StAR) protein in R2C Leydig tumor cells. Inhibition of PKC and phospholipase C resulted in significant decreases in steroid production, phosphorylation of cAMP-responsive element binding (CREB) protein, and Star gene transcription under basal conditions in R2C cells. These observations were corroborated in MA-10 and mLTC-1 Leydig tumor cell lines, in which activation of PKC by phorbol-12-myristate-13-acetate (PMA, 10 nM) increased CREB phosphorylation and total StAR (tot-StAR) protein expression. However, induction of StAR protein by PMA did not result in the expected concomitant increase in steroids because PKC failed to phosphorylate StAR, the biologically active form of the protein. However, in conjunction with PMA, minor increases in PKA activity using submaximal doses of (Bu)2cAMP (0.05-0.1 mM; a concentration range insufficient for induction of StAR), were able to stimulate dramatic increases in both phospho-StAR (P-StAR) and steroid production. Human chorionic gonadotropin stimulation also resulted in a further enhancement in P-StAR and progesterone production when added to PMA-treated MA-10 cells. Similar results for tot-StAR and P-StAR expression were observed in primary cultures of immature rat Leydig cells treated with PMA and submaximal doses of (Bu)2cAMP. In summary, the present study demonstrates that basal activities of both PKC and PKA play important roles in the constitutive steroidogenic characteristics of R2C cells. This study also demonstrates for the first time a role for PMA-induced PKC in StAR protein regulation and the requirement for submaximal doses of cAMP to produce steroids in Leydig cells.

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Electron paramagnetic resonance studies of cytochrome P-450 and adrenal ferredoxin in single whole rat adrenal glands. Effect of corticotropin

Cited by 39 publications

References 19 publications

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

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

Oxidized adrenodoxin acts as a competitive inhibitor of cytochrome P450scc in mitochondria from the human placenta

Involvement of Protein Kinase C and Cyclic Adenosine 3′,5′-Monophosphate-Dependent Kinase in Steroidogenic Acute Regulatory Protein Expression and Steroid Biosynthesis in Leydig Cells1

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