title: Presence of functionally active cytochrome P-450IIE1 in the plasma membrane of rat hepatocytes.
abstract: Recent experiments have described the presence of cytochrome P-450 and certain P-450 isozymes in the plasma membrane of rat liver. Experiments were carried out to evaluate whether cytochrome P-450IIE1 was present in the plasma membrane fraction of livers from control rats and rats treated with 4-methylpyrazole, which induces this isozyme. Using immunofluorescence, fluorescence was detected at the surface of intact hepatocytes that were initially incubated with anti-P-450IIE1 IgG, but not preimmune IgG, followed by incubating with goat antirabbit IgG conjugated with either fluorescein or rhodamine. The fluorescence appeared to be uniformly distributed across the entire surface. Intense intracellular staining could be observed when the hepatocytes were permeabilized by acetone treatment. Similar results were obtained with control hepatocytes; however, the fluorescence intensity was considerably less than that shown by the induced hepatocytes. Hepatocytes isolated from the pericentral zone of the liver acinus displayed more intense fluorescence at the surface than did hepatocytes from the periportal zone. Purified plasma membranes oxidized dimethylnitrosamine to formaldehyde at rates that were 14% to 30% that of the microsomes, which exceeds the 3% contamination of the plasma membranes by microsomes as assessed by glucose-6-phosphatase activity. Immunoblots of the plasma membranes revealed the presence of a single band, whose intensity of staining was 14% to 26% that of the microsomes. Oxidation of dimethylnitrosamine and immunoblot intensity were about twofold greater with plasma membrane fractions from 4-methylpyrazole-treated rats than controls. These results suggest the presence of inducible, functionally active P-450IIE1 in the plasma membrane, which may be of toxicological significance in view of the preferential metabolism of a variety of hepatotoxins and carcinogens and the elevated production of reactive oxygen intermediates by this isozyme.
authors.affiliation: Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
authors.forename: D
authors.initials: D
authors.lastname: Wu
authors.forename: A I
authors.initials: AI
authors.lastname: Cederbaum
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D051381:Rats; D014018:Tissue Distribution
chemical_list: D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D004128:Dimethylnitrosamine