Densities of NADPH-ferrihemoprotein reductase and cytochrome P-450 molecules in the endoplasmic reticulum membrane of rat hepatocytes.

Watanabe, Jun; Asaka, Youko; Fujimoto, Satoshi; Kanamura, Shinsuke

doi:10.1177/41.1.8417111

Cited by 26 publications

(28 citation statements)

References 13 publications

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“…As described in Matherials and Methods, the molar ratio of CPR to CYP3A4 in the two preparations of Baculosomes used in this study was estimated to be around 3.2:1 and 8:1, while the ratio of CPR to total P450 in liver microsomes is known to be 1:10 [57] or even 1:50 [58,59]. Another important difference is that the Baculosomes do not contain cytochrome b 5 .…”

Section: Effect Of Gsh On Homotropic Cooperativity Of O-debenzylationmentioning

confidence: 97%

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Davydov

Davydova

Tsalkova

et al. 2008

Archives of Biochemistry and Biophysics

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Glutathione (GSH) exerted a profound effect on the oxidation of 7-benzyloxy-4-(trifluoromethyl) coumarin (BFC) and 7-benzyloxyquinoline (BQ) by human liver microsomes as well as by CYP3A4-containing insect cell microsomes (Baculosomes). The cooperativity in O-debenzylation of both substrates is eliminated in the presence of 1-4 mM GSH. Addition of GSH also increased the amplitude of the 1-PB induced spin shift with purified CYP3A4 and abolished the cooperativity of 1-PB or BFC binding. Changes in fluorescence of 6-bromoacetyl-2-dimethylaminonaphthalene attached to the cysteine-depleted mutant CYP3A4(C58,C64) suggest a GSH-induced conformational changes in proximity of α-helix A. Importantly, the K S value for formation of the GSH complex and the concentrations in which GSH decreases CYP3A4 cooperativity are consistent with the physiological concentrations of GSH in hepatocytes. Therefore, the allosteric effect of GSH on CYP3A4 may play an important role in regulation of microsomal monooxygenase activity in vivo.

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Section: Effect Of Gsh On Homotropic Cooperativity Of O-debenzylationmentioning

confidence: 97%

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Davydov

Davydova

Tsalkova

et al. 2008

Archives of Biochemistry and Biophysics

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“…The use of detergents to modulate protein-protein interactions provided the first direct evidence for higher order functional complexes. Based on sedimentation and gel filtration studies, P450 1A2 (36) and 2B4 (37) formed pentamers that associated with CPR for optimal activity (P450 5 •CPR). CPR also formed dimers and other higher ordered complexes; however, low amounts of detergent dispersed the complexes indicating CPR homo-oligomers were less stable than those for the P450s.…”

Section: Discussionmentioning

confidence: 99%

“…Although the most notable P450 2E1 substrate is ethanol, pnitrophenol (pNP) is regarded as a typical model substrate (14). The ability to transform these compounds to products depends on the coupling of electron-transfer processes between P450 2E1 and redox partners, CPR and cyt b 5 (14). The mechanism by which the functional complex (es) form for these prospective partners remains to be resolved.…”

mentioning

confidence: 99%

Global Analysis of Protein−Protein Interactions Reveals Multiple CYP2E1−Reductase Complexes

et al. 2007

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Although a single binary functional complex between cytochrome P450 and cytochrome P450 reductase (CPR) has been generally accepted in the literature, this simple model failed to explain experimentally observed catalytic activity of recombinant P450 2E1 in dependence on the total concentration of added CPR-K56Q mutant. Our rejection of the simplest 1:1 binding model was based on two independent lines of experimental evidence. First, under the assumption of the 1:1 binding model, separate analyses of titration curves obtained while varying either P450 or CPR concentrations individually produced contradictory results. Second, an asymmetric Job plot suggested the existence of higher order molecular complexes. To identify the most probable complexation mechanism, we generated a comprehensive data set where the concentrations of both P450 and P450 were varied simultaneously, rather than one at a time. The resulting two-dimensional data were globally fit to 32 candidate mechanistic models, involving the formation of binary, ternary, and quaternary P450:CPR complexes, in the absence or presence or P450 and CPR homodimers. Of the 32 candidate models (mechanisms), two models were approximately equally successful in explaining our experimental data. The first plausible model involves the binary complex P450•CPR, the quaternary complex (P450) 2 • (CPR) 2 , and the homodimer (P450) 2 . The second plausible model additionally involves a weakly bound ternary complex (P450) 2 •CPR. Importantly, only the binary complex P450•CPR seems catalytically active in either of the two most probable mechanisms.Protein-ligand and protein-protein interactions are fundamental to biological processes such as signal transduction, chemical transformations, and electron transport. An understanding of the role of these processes in biological function requires the identification of the detailed interaction mechanisms. These details provide a framework in which to understand how the process of molecular recognition maintains proper homeostasis, or leads to deleterious conditions. In this study, we characterize the details of protein-protein interactions involving cytochrome P450 1 (P450) and the cytochrome P450 reductase (CPR). Traditional experimental protocols and data-analytic methods applied to this two-component system failed to provide a clear answer regarding the binding stoichiometry. We describe an alternative data-analytic approach applicable to any multi-component system. The method is based on a general numerical analysis of simultaneous biochemical equilibria, without any restriction on the number of component molecular species or the number of complexes they form (1). Our results Microsomal P450 enzymes are major catalysts in the oxidative transformation of a structurally diverse class of compounds including steroids, fatty acids, hormones, antibiotics, and a wide variety of artificially produced chemicals (xenobiotics), such as drugs, food additives, and environmental contaminants (2). P450s convert lipid-soluble molecules t...

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“…In the second group (P-450 1A and albumin), a curvilinear relationship was found between immunostaining intensity owing to the amount of antigen immobilized on NC filters, and between the intensity in sections and section thickness. The curvilinear relationship may be attributed to an occurrence of steric hindrance [17], antibody trapping [15], loss of antigen from sections [15] or reduction in antibody binding owing to a high antibody concentration [8]. Therefore, immunostaining intensity resulting from P-450 1A or albumin does not simply reflect the antigen amount in filters or sections.…”

Section: Discussionmentioning

confidence: 99%

“…The content of P-450 reductase in the lysates was determined by enzyme-linked immunosorbent assay [17]. The albumin content was measured with a Rat Albumin SRID kit (Binding Site, Birmingham, UK).…”

Section: Biochemical Methodsmentioning

confidence: 99%

Examination of the Relationship between Immunostaining Intensity and Antigen Amount with an Antigen-immobilized Filter Model System and Sections.

Watanabe

Kanamura

1997

Acta Histochem. Cytochem

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Densities of NADPH-ferrihemoprotein reductase and cytochrome P-450 molecules in the endoplasmic reticulum membrane of rat hepatocytes.

Cited by 26 publications

References 13 publications

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Effect of glutathione on homo- and heterotropic cooperativity in cytochrome P450 3A4

Global Analysis of Protein−Protein Interactions Reveals Multiple CYP2E1−Reductase Complexes

Examination of the Relationship between Immunostaining Intensity and Antigen Amount with an Antigen-immobilized Filter Model System and Sections.

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