[22] The measurement of difference spectra: Application to the cytochromes of microsomes

Estabrook, Ronald W.; Werringloer, J.

doi:10.1016/s0076-6879(78)52024-7

Cited by 257 publications

(117 citation statements)

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“…computer software programme controlled and operated the unit, and collected and plotted the spectral data (Backes & Canady, 1981;Estabrook & Werringloer, 1978). The spectral data were analyzed with the LIGAND programme (Munson & Rodbard, 1980).…”

Section: Methodsmentioning

confidence: 99%

H₃ receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

LaBella

Queen

Glavin

et al. 1992

British J Pharmacology

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Section: Methodsmentioning

confidence: 99%

H₃ receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

LaBella

Queen

Glavin

et al. 1992

British J Pharmacology

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“…The concentration of cyt b 5 in the pellet and supernatant was determined spectrophotometrically after reduction with dithionite using an extinction coefficient of 185 cm Ϫ1 mM Ϫ1 for the absorbance change, which occurs at 426 Ϫ 409 nm (⌬⑀ 426 Ϫ 409 nm ϭ 185 cm Ϫ1 mM Ϫ1 ) when cyt b 5 was reduced by dithionite. The cyt b 5 concentration was corrected for the contribution of cyt P450 in the microsomes (28). 5 is an amphipathic protein, which contains an NH 2 -terminal heme domain anchored to the membrane by a COOH-terminal transmembrane ␣-helix.…”

Section: Stimulation Of Cyt P450 2b4-catalyzed Metabolism Of Methoxyfmentioning

confidence: 99%

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Clarke

Bidwai

et al. 2004

Journal of Biological Chemistry

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Cytochrome b 5 (cyt b 5 ) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane ␣-helix at its carboxyl terminus. These two domains are connected by an ϳ15-amino acid linker domain, Ser 90 -Asp 104 , which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b 5 to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b 5 to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b 5 except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b 5 to interact with cyt P450 2B4. A model for the membranebound cyt b 5 -cyt P450 complex is presented.Microsomal cytochrome b 5 (cyt b 5 ) 1 is an amphipathic electron transfer hemoprotein located in the membrane of the endoplasmic reticulum. It provides electrons for a broad range of reactions, including fatty acid desaturation, cholesterol biosynthesis, and a variety of cytochrome P450-dependent oxidation and hydroxylation reactions (1, 2).Cytochrome P450 (cyt P450) is responsible for the oxidation of a large number of substrates. The overall stoichiometry of the reaction catalyzed by cyt P450 is shown in Reaction 1, where RH is the substrate (3).The cyt P450 catalytic reaction occurs via a reaction cycle that involves substrate binding, reduction of the ferric heme, O 2 binding, reduction of the oxyferrous heme (second electron transfer), substrate oxidation, and finally product dissociation (4). Both ferric and oxyferrous cyt P450 receive electrons from NADPH via its redox partner, NADPH-cytochrome P450 reductase (cyt P450 reductase), whereas cyt b 5 can donate the second electron. Because of the high redox potential of Х20 mV for cyt b 5 , the first electron required to reduce cyt P450 from the ferric to the ferrous state is always transferred from NADPH via cyt P450 reductase. However, as the redox potential of cyt P450 increases from ХϪ230 mV to ϩ50 mV on transition to the oxyferrous state, the second electron can be obtained from either cyt P450 reductase or cyt b 5 (5, 6).Depending on the substrate, isozyme of cyt P450 and the experimental conditions cyt b 5 can stimulate, inhibit, or have no effect on cyt P450 activity (2). Studies of the stoichiometry of the metabolism of benzphetamine and methoxyflurane by cyt P450 2B4 have shown that cyt b 5 increases the efficiency of catalysis by cyt P450 2B4 primarily by decreasing the formation of the side-product superoxide. However, cyt b...

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“…Cytochrome b 5 Concentration-Cytochrome b 5 concentration was determined by recording the reduced minus the oxidized spectrum (absorbance 185 mM Ϫ1 cm Ϫ1 ) as described by Estabrook and Werringloer (22).…”

Section: Analytical Proceduresmentioning

confidence: 99%

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

Clement

Lomb

Möller

1997

Journal of Biological Chemistry

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Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochrome b 5 , its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83-92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxic N-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason why N-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.The metabolism of nitrogen-containing functional groups has become a topic of considerable interest since the early discovery that N-hydroxylated intermediates are often responsible for the toxic and/or carcinogenic properties of aromatic amines, hydrazines, and amides (1). On the other hand, the more facile N-oxygenation of secondary and tertiary alkylamines to hydroxylamines and N-oxides was considered as a route for detoxication (2, 3), and it was generally assumed that the strongly basic nitrogen compounds were metabolically stable. However, we have demonstrated that even the protonated hydrophilic amidines (4 -6), as well as diamidines such as pentamidine and diminazene and also guanidines and amidinohydrazones, are capable of undergoing metabolic N-oxygenation by liver microsomal cytochrome P450 monooxygenases (7,8). The N-oxygenation of these functional groups produces more reactive metabolites, and the genotoxic properties of benzamidoxime are well known (9). During investigations of the metabolic fate of strongly basic N-hydroxylated xenobiotics, we observed that they were readily reduced both in vivo and in vitro by a microsomal system present in all mammalian species (rats, rabbits, pigs, and humans) tested to date (10 -13).Preliminary experiments indicated that this system (10) had many of the characteristics of the microsomal O 2 -insensitive hydroxylamine reductase descri...

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[22] The measurement of difference spectra: Application to the cytochromes of microsomes

Cited by 257 publications

References 7 publications

H₃ receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

H₃ receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

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[22] The measurement of difference spectra: Application to the cytochromes of microsomes

Cited by 257 publications

References 7 publications

H3 receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

H3 receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis.

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

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H₃ receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450

H₃ receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450