Membrane Topology of Guinea Pig Cytochrome P450 17α Revealed by a Combination of Chemical Modifications and Mass Spectrometry
- 13 November 2003
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 42 (49), 14663-14669
- https://doi.org/10.1021/bi035096z
Abstract
Cytochrome P450s in endoplasmic reticulum membranes function in the hydroxylation of exogenous and endogenous hydrophobic substrates concentrated in the membranes. The reactions require electron supplies from NADPH−cytochrome P450 reductase in the same membranes. The membranes play important roles in the reaction of cytochrome P450. The membrane topology of guinea pig P450 17α was investigated on the basis of the differences in reactivity to hydrophilic chemical modification reagents between those in the detergent-solubilized state and proteoliposomes. Recombinant guinea pig cytochrome P450 17α was purified from Escherichia coli and incorporated into liposome membranes. Lysine residues in the detergent-solubilized P450 17α and in the proteoliposomes were acetylated with acetic anhydride at pH 9.0, and the acidic amino acid residues were conjugated with glycinamide at pH 5.0 by the aid of a coupling reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The modifications were performed under conditions where the denatured form, P420, was not induced. The modified P450 17α's were digested by trypsin, and the molecular weights of the peptide fragments were determined by MALDI-TOF mass spectrometry. From the increase in the molecular weights of the peptides, the positions of modifications could be deduced. In the detergent-solubilized state, 11 lysine residues and 7 acidic amino acid residues were modified, among which lysine residues at positions 29, 59, 490, and 492 and acidic residues at 211, 212, and/or 216 were not modified in the proteoliposomes. Both the N- and C-terminal domains and the putative F−G loop were concluded to be in or near the membrane-binding domains of P450 17α.Keywords
This publication has 14 references indexed in Scilit:
- Fluorescence Resonance Energy Transfer Analysis of Cytochromes P450 2C2 and 2E1 Molecular Interactions in Living CellsPublished by Elsevier BV ,2003
- Membrane-Protein Interactions Contribute to Efficient 27-Hydroxylation of Cholesterol by Mitochondrial Cytochrome P450 27A1Published by Elsevier BV ,2002
- Membrane reconstitution of recombinant guinea pig cytochrome P45017α and the effects of site-directed mutagenesis on androgen formationThe Journal of Steroid Biochemistry and Molecular Biology, 2002
- The Rate-determining Step in P450 C21-catalyzing Reactions in a Membrane-reconstituted SystemPublished by Elsevier BV ,2001
- Identification of the Binding Site on Cytochrome P450 2B4 for Cytochrome b 5 and Cytochrome P450 ReductaseJournal of Biological Chemistry, 1998
- Characterization of the Structural Difference between Active and Inactive Forms of the Ras Protein by Chemical Modification Followed by Mass Spectrometric Peptide MappingAnalytical Biochemistry, 1997
- Influence of Matrix Solution Conditions on the MALDI-MS Analysis of Peptides and ProteinsAnalytical Chemistry, 1996
- Two modes of binding of adrenal NADPH–cytochrome P-450 reductase to liposomal membranesBiochimica et Biophysica Acta (BBA) - Biomembranes, 1987
- High-resolution crystal structure of cytochrome P450camJournal of Molecular Biology, 1987
- Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies.The Journal of cell biology, 1987