Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum.
Recent cumulative evidence suggests that liver microsomal cytochrome P-450 (P-450) is exposed to the cytosol with the exception of the N-terminal peptide (amino acid residues 1 to 21), or two peptides (residues 1 to 60). We tested the localization of the N-terminal methionine residue of P-450IIB1 of rat liver microsomes in the natural membrane with the site-specific reagent fluorescein isothiocyanate. The N-terminus of isolated P-450 was stoichiometrically modified in solution with fluorescein isothiocyanate. In intact microsomes, the N-terminus was not modified but became accessible to the reagent when the membrane was dissolved with Triton X-100. Our results indicate that the N-terminus faces the lumen of the endoplasmic reticulum, and we propose that P-450 spans the membrane only once with amino acid residues 1 to 21.
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Laboratorium für Biochemie, Eidgenössische Technische Hochschule, ETH-Zentrum, Zürich, Switzerland.
Winterhalter
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KH
Richter
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D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006900:Hydroxylation; D008297:Male; D008715:Methionine / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D013501:Surface-Active Agents; D013861:Thiocyanates; D014357:Trypsin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013501:Surface-Active Agents; D013861:Thiocyanates; D011092:Polyethylene Glycols; D017830:Octoxynol; D003577:Cytochrome P-450 Enzyme System; D008715:Methionine; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate