Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
Expression of cytochrome P450 2A6 in Escherichia coli: Purification, spectral and catalytic characterization, and preparation of polyclonal antibodies
Autore:
Soucek, P;
Indirizzi:
Natl Publ Hlth Inst, Ctr Occupat Dis, Biotransformat Grp, Prague 10042, Czech Republic Natl Publ Hlth Inst Prague Czech Republic 10042 ue 10042, Czech Republic
Titolo Testata:
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
fascicolo: 2, volume: 370, anno: 1999,
pagine: 190 - 200
SICI:
0003-9861(19991015)370:2<190:EOCP2I>2.0.ZU;2-D
Fonte:
ISI
Lingua:
ENG
Soggetto:
HUMAN LIVER-MICROSOMES; COUMARIN 7-HYDROXYLATION; SACCHAROMYCES-CEREVISIAE; METABOLIC-ACTIVATION; POLYACRYLAMIDE GELS; PROTEINS; IDENTIFICATION; RECONSTITUTION; REDUCTASE; OXIDATION;
Keywords:
CYP2A6; heterologous expression; protein purification; catalytic activity; coumarin; antibodies;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
50
Recensione:
Indirizzi per estratti:
Indirizzo: Soucek, P Natl Publ Hlth Inst, Ctr Occupat Dis, Biotransformat Grp, Srobarova 48, Prague 10042, Czech Republic Natl Publ Hlth Inst Srobarova 48 Prague Czech Republic 10042 ic
Citazione:
P. Soucek, "Expression of cytochrome P450 2A6 in Escherichia coli: Purification, spectral and catalytic characterization, and preparation of polyclonal antibodies", ARCH BIOCH, 370(2), 1999, pp. 190-200

Abstract

Cytochrome P450 (CYP) 2A6 is the principal human enzyme catalyzing coumarin 7-hydroxylation and is known to be involved in the metabolism of halothane, nicotine, and metabolic activation of butadiene and nitrosamines, In this paper expression of CYP2A6 in Escherichia coli is reported. In order to achieve expression, the N-terminus of protein was modified by PCR mutagenesis. The N-terminal variant with only a single amino acid change showed expression of 210 nmol of CYP2A6/liter of culture, Recombinant CYP2A6 protein was purified to electrophoretic homogeneity and further characterized. Absolute spectra were typical for CYP proteins and indicated low spin characteristics of isolated protein. Due to a hydrophobic segment the N-terminal aminoacid sequence of recombinant CYP2A6 was blocked. The N-terminal formyl-methionine block was removed by mild acid treatment. Purified CYP2A6 had good catalytic activity toward marker substrate coumarin in a reconstituted system (K-m = 1.48 +/- 0.37 mu M, V-max = 3.36 +/- 0.18 nmol product/min/nmol CYP). Its activity in the reconstituted system was stimulated by the presence of cytochrome b(5) and glutathione. CYP2A6 was shown to metabolize chlorzoxazone in the reconstituted system with activity of 0.32 nmol of product/min/nmol of CYP, and thus caution should be taken when interpretation of CYP2E1 in vivo phenotyping data is performed. Rabbit polyclonal antibodies were produced against recombinant CYP2A6 and proved to be very useful for immunoblotting and immunoinhibition studies. Availability of this expression system and specific antibodies should facilitate characterization of the roleof CYP2A6 the metabolism of chemicals and in the study biological relevance of genetic polymorphisms of C his enzyme. (C)1999 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 03/07/20 alle ore 00:28:01