Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosphates.
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Phosphate and pyrophosphate increased the rate of reduction of adrenodoxin by NADPH-adrenodoxin reductase and NADPH, pyrophosphate being one order more effective than the former. However, the cytochrome c reduction by the electron transport system was inhibited in the presence of inorganic (pyro)phosphate. On the other hand, ADP and ATP enhanced the rates of reduction of both adrenodoxin and cytochrome c through adrenodoxin by the electron transport system. GTP also enhanced the rate of reduction of cytochrome c by this system, whereas AMP showed no appreciable enhancement. These inorganic and nucleotide phosphates did not affect the rate of ferricyanide reduction by the reductase. (+info)
A tricistronic human adrenodoxin reductase-adrenodoxin-cytochrome P450 27A1 vector system for substrate hydroxylation in Escherichia coli.
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Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells.
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After ovulation of an oocyte, granulosa cells of the ovarian follicle differentiate into luteal cells and become a major factor dedicated to the synthesis of the steroid hormone progesterone. We recently established granulosa cell lines by cotransfection of granulosa cells with SV-40 and Ha-ras oncogene. In these cells progesterone secretion can be induced by cAMP as in normal rat granulosa cells. The induction of progesterone secretion is observed only after approximately 24 h and closely follows the delayed but quantitatively dramatic induction of the mitochondrial cytochrome P450scc which catalyzes the first step in steroid hormone biosynthesis. The mitochondrial P450 system electron transport proteins, adrenodoxin and adrenodoxin reductase, are also induced but adrenodoxin shows a faster induction. Immunofluorescence studies show that the three enzymes are induced in all cells and incorporated into all mitochondria uniformly. Electron microscopic examination using immunogold technique further confirms this and reveals that adrenodoxin is predominantly located on the matrix side of the inner mitochondrial membrane. Thus, adrenodoxin, which is a small highly charged protein, shows a distribution similar to P450scc which is an integral membrane protein. The uniformity of the response of the cells provides further evidence for the homogeneity of the cell line and makes this new granulosa cell line a highly promising system for the study of the molecular mechanisms involved in changes in gene expression during the process of granulosa cell differentiation. (+info)
Mechanism of corticotropin and cAMP induction of mitochondrial cytochrome P450 system enzymes in adrenal cortex cells.
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We studied the kinetics of corticotropin (ACTH) induction of mitochondrial cytochromes P450scc and P450c11 and their electron transport proteins, adrenodoxin and adrenodoxin reductase, in bovine adrenal cortex cells in primary culture. The mRNA levels of these enzymes increase and reach a peak within 3-12 h after ACTH addition. The protein levels of adrenodoxin reductase and P450scc show an increase only nearly 24 h after ACTH addition. After ACTH addition, the intracellular level of cAMP reaches maximal levels within 5 min, and then decreases gradually over 60 min. Hence, we examined the effect of a pulse of ACTH or cAMP analogs on enzyme and mRNA levels. Exposure of the cells to ACTH for 1-2 h was sufficient for maximal induction of the enzymes and P450scc mRNA. In contrast, the induction of the enzymes and the mRNA by cAMP analogs or forskolin required the continuous presence of these agents for over 12 h. But, these agents stimulated cortisol secretion to the medium quickly, indicating that they can activate some intracellular processes while not showing any effect on enzyme induction. The absence of any effect of prolonged cAMP pulses on enzyme and mRNA levels weakens the previous hypothesis that cAMP is the sole second messenger for the ACTH induction of steroidogenic enzymes in adrenal cortex cells. The inductive ability of a brief pulse of ACTH indicates that ACTH can rapidly initiate a series of reactions that result in enzyme induction many hours later. (+info)
Coenzyme Q biosynthesis: Coq6 is required for the C5-hydroxylation reaction and substrate analogs rescue Coq6 deficiency.
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Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc.
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Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum. (+info)
Ionic effects on adrenal steroidogenic electron transport. The role of adrenodoxin as an electron shuttle.
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We have shown (Seybert, D., Lambeth, D., and Kamin, H. (1978), J. Biol. Chem. 253, 8355-8358) that, whereas the 1:1 complex between adrenodoxin reductase and adrenodoxin is the active species for cytochrome c reduction, the complex is not sufficient to allow cytochrome P-45011 beta-mediated hydroxylations;adrenodoxin in excess of reductase is required. In the present studies, reduction by NADPH of excess adrenodoxin is shown to occur at a rate sufficient to support both cytochrome P-450 11 beta-mediated hydroxylation of deoxycorticosterone, and cytochrome P-450sec-mediated side chain cleavage of cholesterol. Oxidation-reduction potential and ion effect studies indicate that the mechanism of steroidogenic electron transport involves an adrenodoxin electron "shuttle" rather than a macromolecular complex of reductase, adrenodoxin, and cytochrome. The oxidation-reduction potential of adrenodoxin is shifted about -100 mV when bound to reductase, and reduction of the iron-sulfur protein thus promotes dissociation of the complex. The rate of adrenodoxin reduction is first stimulated, then inhibited by increasing salt; the effect is ion-specific, with Ca2+ approximately Mg2+ greater than Na+ greater than NH/+. Similar ion-specific rate effects are observed for both of the cytochrome P-450-mediated hydroxylations, indicating that the same reduction mechanism is required for these reactions. Increasing salt concentrations caused dissociation of the complex; dissociation of the form of the complex containing reduced adrenodoxin occurred at lower salt concentrations than that containing oxidized adrenodoxin. The order of effectiveness of ions in causing dissociation is the same as the order for stimulation of adrenodoxin reduction, suggesting a dissociation step in the mechanism. This proposed model, together with dissociation constants for the form of the complex containing either oxidized or reduced adrenodoxin, allows accurate prediction of the salt rate effects curve. For all ions, an activity maximum is seen at the ion concentration which produces the largest molar difference between associated-oxidized and dissociated-reduced states, and the model predicts the positions of the maxima for adrenodoxin reduction, 11 beta-hydroxylation, and side chain cleavage. Thus reduction-induced dissociation of adrenodoxin from adrenodoxin reductase appears to be a required step in steroidogenic electron transport by this system, and a role for adrenodoxin as a mobile electron shuttle is proposed. (+info)