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

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F14%3A00507239" target="_blank" >RIV/61388971:_____/14:00507239 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/14:10286787

Výsledek na webu

<a href="http://www.nel.edu/userfiles/articlesnew/1520711919_35_s2_jecmen_114-122-pdf.pdf" target="_blank" >http://www.nel.edu/userfiles/articlesnew/1520711919_35_s2_jecmen_114-122-pdf.pdf</a>

DOI - Digital Object Identifier

—

Jazyk výsledku

angličtina

Název v původním jazyce

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

Popis výsledku v původním jazyce

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. nThe 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.

Název v anglickém jazyce

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

Popis výsledku anglicky

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. nThe 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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

<a href="/cs/project/LO1509" target="_blank" >LO1509: Pražská infrastruktura pro strukturní biologii a metabolomiku II</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2014

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Neuroendocrinology Letters

ISSN

0172-780X

e-ISSN

—

Svazek periodika

35

Číslo periodika v rámci svazku

2

Stát vydavatele periodika

LU - Lucemburské velkovévodství

Počet stran výsledku

9

Strana od-do

114-122

Kód UT WoS článku

000351064900015

EID výsledku v databázi Scopus

2-s2.0-84928995722