Spectrophotometric properties of a triton-clarified steroid 21-Hydroxylase system of adrenocortical microsomes

Narasimhulu, Shakunthala; Cooper, David Y.; Rosenthal, Otto

doi:10.1016/0024-3205(65)90328-0

Cited by 120 publications

(21 citation statements)

References 8 publications

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“…On the other hand, requirements for NADPH and oxygen are not unique for these processes, since hydrogen peroxide formation by NADPH oxidase or cytochrome P-450 also needs NADPH and oxygen and the latter process is sensitive to carbon monoxide. Moreover, although ethanol gives a binding spectrum with cytochrome P-450 [32], this does not necessarily indicate that it is a substrate, since non-specific binding has been observed [33,41]. Therefore, we conclude that no convincing evidence exists that microsomal ethanol oxidation and drug metabolism occur via similar mechanisms.…”

Section: Is Microsomal Nadph-dependent Ethanol Oxidation a Mixed Funcmentioning

confidence: 64%

Hepatic Microsomal Ethanol Oxidation

Thurman

Ley²,

Scholz³

1972

European Journal of Biochemistry

579

192

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Microsomes from rat liver form hydrogen peroxide in the presence of an NADPH-generating system in proportion to protein concentrations as determined by three independent methods : ferrithiocyanate, cytochrome c peroxidase, and scopoletin fluorescence. Maximal rates observed were about 15 pmol H,O,/g microsomal protein per minute. The oxygen concentration for half-maximal rates was 50 pM. It is suggested that NADPH-dependent hydrogen peroxide formation in microsomes is mainly due to NADPH oxidase; however, partial inhibition by carbon monoxide suggests that about one third arises from the autoxidation of cytochrome P-450.Similarities exist between microsomal acetaldehyde production from ethanol (i.e. the microsomal ethanol-oxidizing system of Lieber and DeCarli [4]) and hydrogen peroxide formation : viz. requirement for NADPH and oxygen, identical oxygen concentrations for halfmaximal rates, and sensitivity to carbon monoxide. Microsomal acetaldehyde production in the presence of either an NADPH-or an H,O,-generating system exhibits identical characteristics as follows: (a) ethanol concentration for half-maximal rates (i.e. 12 mM); (b) dependency of maximal rates on rates of hydrogen peroxide formation; (c) competitive inhibition by peroxidatic substrates for catalase, e.g. formate (half-maximal effect: 150 mM); (d) inhibition by catalase inhibitors, e.g. azide (half-maximal effect : 50 pM), with identical azide insensitive rates ; (e) diminished acetaldehyde production in microsomes from rats pretreated with aminotriazole or pyrazole with identical residual rates. Moreover, NADPH-dependent acetaldehyde production is suppressed in the presence of an active H,O,-utilizing system. Thus, it is concluded that the NADPH-dependent microsomal ethanol-oxidizing system of Lieber and DeCarli [4] is due to a hydrogen peroxide formation from NADPH and a subsequent peroxidation of ethanol by contaminating catalase. The data indicate that the existence of a unique system in addition to the peroxidatic reaction of catalase as postulated recently [4] is highly doubtful.

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Section: Is Microsomal Nadph-dependent Ethanol Oxidation a Mixed Funcmentioning

confidence: 64%

Hepatic Microsomal Ethanol Oxidation

Thurman

Ley²,

Scholz³

1972

European Journal of Biochemistry

579

192

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“…Furthermore, the P45017a enzyme present in these membranes is not rendered soluble by treatment with 0.1 M sodium carbonate (pH 11.5), indicating that it is an integral membrane protein (22) (23). Binding of substrates was also observed by detection of substrate-induced difference spectra (24) in intact bacteria after addition of the C21 steroids pregnenolone, progesterone, 17a-hydroxypregnenolone, or 17a-hydroxyprogesterone (Fig. 2B).…”

Section: Resultsmentioning

confidence: 88%

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

Barnes

Arlotto

Waterman

1991

Proc. Natl. Acad. Sci. U.S.A.

520

355

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When the cDNA encoding bovine microsomal 17a-hydroxylase cytochrome P450 (P45017a) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017a per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017a can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017a enzyme. Surprisingly, it is found that E. coli contain an electrontransport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17a-hydroxylase and 17,20-lyase activities of P45017a. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structurefunction analysis of the cytochrome P450 system. Microsomal cytochromes P450 are integral membrane hemoproteins that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous compounds. Deriving reducing equivalents from NADPH via a membrane-bound flavoprotein oxidoreductase (NADPH-cytochrome P450 reductase), these mixed-function oxidases activate molecular oxygen so as to insert one atom into a lipophilic substrate and the other atom into water. Recent study of the molecular aspects underlying eukaryotic cytochrome P450 structure and function has relied on the techniques of molecular biology to synthesize specific individual forms ofcytochrome P450 in heterologous expression systems. Yeast (1), COS 1 (2), and eukaryotic cells infected with a viral vector (3, 4) have been used as hosts for the heterologous expression of cytochrome P450 molecules; however, each has limitations to their usefulness as systems for structure-function analysis. Although the bacterium Escherichia coli has demonstrated great usefulness in the expression of many prokaryotic and eukaryotic proteins, E. coli as an expression system for cytochrome P450 has been limited primarily to the soluble prokaryotic forms of this gene superfamily (5). We have used the cDNA encoding bovine 17a-hydroxylase cytochrome P450 (P45017a) to examine the utility of E. coli as an expression system for eukaryotic cytochromes P450 in the hopes that such a system might prove suitable for both enzymatic and structural studies. This microsomal cytochrome P450 catalyzes the regiospecific and stereospecific 17a-hydroxylation of the C21 steroids pregnenolone and progesterone in the pathway leading to the production of cortisol and the 17,20-lyase conversion of 17a-hydroxypregnenolone to the C19 adrenal androgen dehydroepiandrosterone (DHEA) in the adrenal cortex of most mammalian species. P45017a also converts these 17a-hydroxylat...

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“…The addition of FAD and FMN enhances activity, whereas, cytochrome c, which acts as an alternate acceptor of electrons from the P450 reductase, inhibits sabinene hydroxylation. All of these features are typical of cytochrome P450 hydroxylases (Omura and Sato 1964;Estabrook et al 1963;Narasimhulu et al 1965;Testa and Jenner 1981).…”

Section: Other Monoterpene Hydroxylasesmentioning

confidence: 95%

Cytochrome P450 oxygenases of monoterpene metabolism

Mau

Croteau

2006

Phytochem Rev

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The cytochrome P450 monoterpene oxygenases are largely responsible for imparting structural and functional diversity to this family of natural products. In most cases, cytochrome P450-mediated allylic hydroxylation of a parental monoterpene olefin leads to a series of redox transformations and conjugation reactions which yield a family of structurally related derivatives and isomers. An overview is provided of the extant monoterpene oxygenases, with examples mainly from the mint (Lamiaceae) family of essential oil plants, and, where possible, information on the structure, mechanism, localization and regulation of these enzymes is described. The review concludes with a brief assessment of biotechnological applications and a view to future research in this area.

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Spectrophotometric properties of a triton-clarified steroid 21-Hydroxylase system of adrenocortical microsomes

Cited by 120 publications

References 8 publications

Hepatic Microsomal Ethanol Oxidation

Hepatic Microsomal Ethanol Oxidation

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

Cytochrome P450 oxygenases of monoterpene metabolism

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