Quantification of interactions between cytochrome P450 2B4 and cytochrome b(5) in a functional membrane complex

0507239 - MBÚ 2020 RIV LU eng J - Journal Article
Ječmen, Tomáš - Ptáčková, Renata - Kavan, Daniel - Černá, V. - Hodek, P. - Stiborová, M. - Hudeček, J. - Šulc, Miroslav
Quantification of interactions between cytochrome P450 2B4 and cytochrome b(5) in a functional membrane complex.
Neuroendocrinology Letters. Roč. 35, č. 2 (2014), s. 114-122. ISSN 0172-780X. E-ISSN 2354-4716
R&D Projects: GA ČR(CZ) GAP207/12/0627; GA MŠMT(CZ) LO1509
Institutional support: RVO:61388971
Keywords : cytochrome P450 2B4 * cytochrome b(5) * photo-initiated cross-linking
OECD category: Microbiology
Impact factor: 0.799, year: 2014
Method of publishing: Open access
http://www.nel.edu/userfiles/articlesnew/1520711919_35_s2_jecmen_114-122-pdf.pdf

The mammalian mixed function oxidase (MFO) system participates in hydroxylation of many hydrophobic endogenous compounds as well as xeno-biotics such as drugs and carcinogens. This biotransformation system, located in a membrane of endoplasmic reticulum, consists of cytochrome P-450 (P450), NADPH: P450 oxidoreductase and a facultative component, cytochrome b(5). The knowledge of the interactions among the individual components of the MFO system is essential to understand the relationships between the structure and function of this system that finally dictate a qualitative and quantitative pattern of produced metabolites (e.g. detoxified xenobiotics and/or activated carcinogens). To elucidate the quantitative aspects of the interactions within the MFO system we acquired the photo-initiated cross-linking approach.
The photo-initiated cross-linking employing cytochrome b5 as a protein nanoprobe [an amino acid analogue of methionine (pMet) was incorporated into cytochrome b5 sequence during recombinant expression] was used to quantify its interaction with P450 2B4 in a functional membrane complex. The cross-linking was initiated by UV-irradiation that formed from a pMet photolabile diazirine group highly reactive carbene biradical. This biradical is able to covalently bind amino acids in the close proximity and to form cross-link. The Met 96 of cytochrome b5 is situated in a linker region between its catalytic and membrane domains, while Met 126 and 131 are located in its membrane domain. The combination of several methods (electrophoresis in polyacrylamide gel, isoelectric focusing, Edman N-terminal degradation and amino acid analysis) was employed to characterize the molar ratio of The successfully produced cytochrome b5 nanoprobe (with confirmed pMet incorporation by mass spectrometry) stimulates the catalytical activity of P450 2B4 when reconstituted with NADPH: P450 oxidoreductase in vitro in dilauroylphosphatidylcholine (DLPC) vesicles.
Permanent Link: http://hdl.handle.net/11104/0298272