title: Immobilization and activity assay of cytochrome P450 on patterned lipid membranes.
abstract: We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.
authors.affiliation: Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ueda
authors.forename: Kenichi
authors.initials: K
authors.lastname: Morigaki
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Tatsu
authors.forename: Noboru
authors.initials: N
authors.lastname: Yumoto
authors.forename: Hiromasa
authors.initials: H
authors.lastname: Imaishi
mesh_terms: D000478:Alkylation; D000818:Animals; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis* / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000032:analysis* / Q000378:metabolism*; D008051:Lipid Bilayers; D051381:Rats
chemical_list: D004800:Enzymes, Immobilized; D008051:Lipid Bilayers; D019363:Cytochrome P-450 CYP1A1