Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate‐adrenodoxin reductase of pig adrenocortical mitochondria

Hiwatashi, Atsuo; Ichikawa, Yoshiyuki; Yamano, Takeshi

doi:10.1016/0014-5793(77)80584-x

Cited by 32 publications

(8 citation statements)

References 21 publications

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“…Adrenodoxin and NADPH -adrenodoxin reductase were purified from bovine adrenal cortex according to the methods described previously (Suhara et al, 1972a, b) with some modifications (Hiwatashi et al, 1977;Lombard0 et al, 1986). P-450,,, preparations separated by 8.5 % SDSPAGE (Laemmli et al, 1970) were transferred to a nitrocellulose filter according to Towbin et al (1979).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Delorme¹,

Piffeteau²,

Viger³

et al. 1995

European Journal of Biochemistry

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The last step of aldosterone biosynthesis, an 1 1p-hydroxylation followed by two 18-hydroxyIations, are catalyzed, in the bovine system, by the same enzyme, the cytochrome P-45Oll, (deoxycorticosterone (DOC) -corticosterone -18-hydroxycorticosterone -aldosterone). The 1 lp-and 18-hydroxylase activities were studied separately with a reconstituted enzymic system, using 1 l-de~xy Steroid Biochem. 33, 119-1241, were characterized for both activities (llp-and 18-hydroxylase). The value of reversible K, for the 18-hydroxylation (Ki = 5 pM for 18-vinylprogesterone and 30 pM for 18-ethynylprogesterone) is lower than that for the 1lp-hydroxylation (30 pM and 100-150 pM, respectively); the former inhibitor is stronger than the latter for both steps.The binding of substrates and inhibitors to the active site was also examined by difference absorption spectroscopy. 18-Vinylprogesterone gave rise to a type I spectrum with a K, value of 35 pM close to that of progesterone, while 18-ethynylprogesterone showed a reverse type I spectrum with a much higher K, value (140 pM). Based on these results, a hypothetical model, involving a conformational change of the enzyme for the second step, is proposed.Keywords: cytochrome ; P-45OI1,; mechanism-based inhibitors ; aldosterone biosynthesis ; 18-vinylprogesterone.Aldosterone overproduction leads to oedematous diseases and hypertension (Corvol et al., 1990). These disorders are clinically treated with spirolactones which act as antagonists at the receptor level. We developed a new approach namely by focusing on the inhibition of cytochrome P-4501,, (P-450,,,), the last enzyme in aldosterone biosynthesis. For this purpose, progesterone derivatives designed as mechanism-based inhibitors of P-4501,, were synthesized in our laboratory (Viger et al., 1988).The most potent inhibitors, tested with rat adrenal cell-free extracts, were 18-vinylprogesterone (1 8-VP) and 18-ethynylprogesterone (18-EP) (Fig. 1) which completely inhibit aldosterone production at 0.8 pM and 8 pM, respectively (Viger et al., 1989). In this study the inhibitions by 18-VP and 18-EP were studied more accurately using a bovine reconstituted enzymic P-450,,,, P-450,,, and P-450,,,,, cytochromes P-450 1 lP-18-hydroxylase, P-450 cholesterol side-chain cleavage, P-450 17a-hydro~ylase/C,,,~, lyase and P-450 aromatase.Enzyme. Steroid 1 ID-monoxygenase; cytochrome P-450, ,8 (EC 1.14.15.4). system including P-45011,, adrenodoxin and adrenodoxin reductase.P-450,,, purified from bovine adrenal cortex can catalyze the 1 lp-hydroxylation of 11 -deoxycorticosterone, the 18-hydroxylation of corticosterone as well as aldosterone formation (Fig. 2) (Wada et al., 1985;Yanagibashi et al., 1986). However two distinct forms of P-45Ol,,, encoded by two different genes exist in the bovine adrenal cortex (for a review, see Muller, 1993). Indeed two bovine P-45011, cDNAs, pc P-450 118-3 and pc P-450 llp-2 were cloned (Moroashi et al., 1987;Kirita et al., 1988). Expression into COS-7 cells revealed that both enzymes were capable of catalyzin...

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Section: Methodsmentioning

confidence: 99%

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Delorme¹,

Piffeteau²,

Viger³

et al. 1995

European Journal of Biochemistry

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“…Adrenodoxin was prepared from bovine adrenals according to the method described by Suhara et al (12), and NADPH-adrenodoxin reductase was prepared by a modified method of Suhara et al (13,14). 25-Hy-droxyvitamin D 3 , and 1␣,25-dihydroxyvitamin D 3 were purchased from Duphar (Weesp, The Netherlands).…”

Section: Methodsmentioning

confidence: 99%

Assay of 25-Hydroxyvitamin D31α-Hydroxylase in Rat Kidney Mitochondria

Eto

Nakamura

Taniguchi

et al. 1998

Analytical Biochemistry

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“…The amino acid residue compositions of the native and pyridoxal-P-modified FMOs were determined with a Hitachi amino acid analyzer, model 835 [23]. In the case of the pyridoxal-P-modified FMO, 18 pM FMO in 10 mM potassium phosphate pH 7.4 was incubated with 2 mM pyridoxal-P in 1 ml for 2 h at 4°C on a shaker.…”

Section: Methodsmentioning

confidence: 99%

An Essential Lysyl Residue (Lys208) in the Substrate-Binding Site of Porcine FAD-Containing Monooxygenase

Ichikawa

1995

Eur J Biochem

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The substrate (amine)-binding site of porcine FAD-containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5'-phosphate (pyridoxal-P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal-P. Upon reduction of pyridoxal-P-treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal-P appeared at 325 nm. The amino acid residue compositions of the native and pyridoxal-P-treated FMOs indicated that the lysyl residues were modified by pyridoxal-P. The about 74% inactivation of the enzymic activity on covalent pyridoxal-P treatment of the FMO was nearly completely prevented in the presence of the substrate, N,N-dimethylaniline. The FMO covalently modified with pyridoxal-P in the presence or absence of N,N-dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse-phase high-performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal-P and was detected at 325 nm in the absence of N,N-dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186-208. These results suggest that Lys208 plays an important role in the substrate (amine)-binding site of FMO.

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Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate‐adrenodoxin reductase of pig adrenocortical mitochondria

Cited by 32 publications

References 21 publications

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Assay of 25-Hydroxyvitamin D31α-Hydroxylase in Rat Kidney Mitochondria

An Essential Lysyl Residue (Lys208) in the Substrate-Binding Site of Porcine FAD-Containing Monooxygenase

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Crystallization and properties of reduced nicotinamide adenine dinucleotide phosphate‐adrenodoxin reductase of pig adrenocortical mitochondria

Cited by 32 publications

References 21 publications

Inhibition of Bovine Cytochrome P‐45011β by 18‐Unsaturated Progesterone Derivatives

Inhibition of Bovine Cytochrome P‐45011β by 18‐Unsaturated Progesterone Derivatives

Assay of 25-Hydroxyvitamin D31α-Hydroxylase in Rat Kidney Mitochondria

An Essential Lysyl Residue (Lys208) in the Substrate-Binding Site of Porcine FAD-Containing Monooxygenase

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Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives

Inhibition of Bovine Cytochrome P‐450_11β by 18‐Unsaturated Progesterone Derivatives