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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:
 [1];  [2];  [3];  [4];  [4];  [5]
  1. Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501 (Japan)
  2. (AIST), Midorigaoka, Ikeda 563-8577 (Japan)
  3. Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda 563-8577 (Japan). E-mail: morigaki-kenichi@aist.go.jp
  4. Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda 563-8577 (Japan)
  5. Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501 (Japan). E-mail: himaish@kobe-u.ac.jp
Publication Date:
OSTI Identifier:
20979882
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 355; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2007.02.058; PII: S0006-291X(07)00327-0; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADSORPTION; BIOASSAY; CELL MEMBRANES; LIPIDS; MICROSOMES; OPTIMIZATION; RATS; SUBSTRATES

Citation Formats

Ueda, Yoshihiro, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Morigaki, Kenichi, Tatsu, Yoshiro, Yumoto, Noboru, and Imaishi, Hiromasa. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.02.058.
Ueda, Yoshihiro, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Morigaki, Kenichi, Tatsu, Yoshiro, Yumoto, Noboru, & Imaishi, Hiromasa. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes. United States. doi:10.1016/j.bbrc.2007.02.058.
Ueda, Yoshihiro, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, Morigaki, Kenichi, Tatsu, Yoshiro, Yumoto, Noboru, and Imaishi, Hiromasa. Fri . "Immobilization and activity assay of cytochrome P450 on patterned lipid membranes". United States. doi:10.1016/j.bbrc.2007.02.058.
@article{osti_20979882,
title = {Immobilization and activity assay of cytochrome P450 on patterned lipid membranes},
author = {Ueda, Yoshihiro and Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology and Morigaki, Kenichi and Tatsu, Yoshiro and Yumoto, Noboru and Imaishi, Hiromasa},
abstractNote = {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.},
doi = {10.1016/j.bbrc.2007.02.058},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 355,
place = {United States},
year = {Fri Apr 20 00:00:00 EDT 2007},
month = {Fri Apr 20 00:00:00 EDT 2007}
}