Membrane integration of recombinant human P450 forms

Shukla, A., Huang, W., Depaz, I. M. and Gillam, E. M. J. (2009) Membrane integration of recombinant human P450 forms. Xenobiotica, 39 7: 495-507. doi:10.1080/00498250902934884


Author Shukla, A.
Huang, W.
Depaz, I. M.
Gillam, E. M. J.
Title Membrane integration of recombinant human P450 forms
Journal name Xenobiotica   Check publisher's open access policy
ISSN 0049-8254
Publication date 2009-07
Year available 2009
Sub-type Article (original research)
DOI 10.1080/00498250902934884
Open Access Status
Volume 39
Issue 7
Start page 495
End page 507
Total pages 13
Editor C. Loannides
Place of publication United Kingdom
Publisher Informa Healthcare
Collection year 2010
Language eng
Subject C1
970106 Expanding Knowledge in the Biological Sciences
060602 Animal Physiology - Cell
060111 Signal Transduction
110307 Gastroenterology and Hepatology
111601 Cell Physiology
Abstract 1. Amino terminal sequence modification of cytochrome P450 enzymes is often necessary to achieve expression in bacteria. The aim of this study was to examine the effect of such modifications on membrane integration and P450 activity. 2. Forms that retained substantial N-terminal hydrophobic sequences remained unaffected by treatments to remove peripheral membrane proteins and were released only by detergent. Truncated P450s 2A13, 2C9 (Delta 3-20),2C19 (Delta 3-20),2D6 (DB11) and 2E1 remained principally membrane-bound, but some P450 was found in the soluble fraction and could be partially extracted by alkaline and high salt treatments. 3. The subcellular localization of P450s 2C9 and 2C19 assessed by fluorescence microscopy mirrored the distribution between subcellular fractions. The MALLLAVFL modified forms of P450 2C9 YFP, P450 2C18 YFP and P450 2C19 YFP were found primarily at the periphery of the cells, whereas the truncated forms of P450 2C9 (Delta 3-20) YFP and 2C19 (Delta 3-20) YFP were observed at the periphery as well as inside the cells. 4. N-terminal variants of P450s 2C9 and 2C19 showed altered kinetics towards form-selective substrates. Rates of diclofenac 4'-hydroxylation by P450 2C9 and luciferin H-EGE metabolism by P450 2C19 were higher for the MALLLAVFL-modified forms compared with the (Delta 3-20) truncated forms despite supplementation of truncated form incubations with additional reductase. 5. Thus, N-terminal sequence modifications changed the degree of membrane integration, potentially affecting subcellular localization, interactions with redox partners, and hence enzymatic activity.
Keyword Recombinant P450s
membrane integration
subcellular localization
membrane proteins
heterologous expression
MECHANISM-BASED INACTIVATION
HUMAN CYTOCHROME-P450 1A2
ESCHERICHIA-COLI
ENDOPLASMIC-RETICULUM
SPECTRAL CHARACTERIZATION
CATALYTIC-ACTIVITY
CHOLESTEROL 7-ALPHA-HYDROXYLASE
EXPRESSION LEVELS
HUMAN LIVER
PURIFICATION
Q-Index Code C1
Q-Index Status Confirmed Code

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
School of Biomedical Sciences Publications
 
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Citation counts: TR Web of Science Citation Count  Cited 8 times in Thomson Reuters Web of Science Article | Citations
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Created: Mon, 07 Sep 2009, 09:35:34 EST by Cameron Harris on behalf of School of Biomedical Sciences