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Presence of functional cytochrome P-450 on isolated rat hepatocyte plasma membrane
I103
rences Schmidt,B., 1963,Am. J. Ophthalmol.55, 208. Okun, E., Gouras,P., 1963,Arch. Ophthalmol.69, 59. I0.tu.14.6 1
on isolated rat Loeper *, J., Desoatoire *, V., Maurice *, M., Beaune * *, P., Feldmann *, G., Larrey *, D. and Pessayre *, D. * Unit&de Recherches de Physiopathologie
HPpatique (INSERM U-24) and * * INSERM Paris, France
U-75, CHU Necker-Enfants-Malades,
Antibodies against cytochrame P-450 are found in some children with autoimmune hepatitis (anti-k& directed against cytochrome P-450 IID6) or in patients with ticrynafen hepatitis (anti-LKM,, directed against cytochrome P-450 IICIO). For such antibodies (or sensitized cytotoxic T cells) to possibly damage the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of the hepatocytes. While this presence has been reported by some authors (Stasieclciand Oesch, 1980), other authors have concluded to its absence. Aim of tb.e study. In view of the conflicting data of the literature on this important topic, we have reinvestigated the possible presence of cytochrome P-450 on the plasma membrane, by both biochemical and morphological methods. Methods. Biochemical studies were performed with a plasma membrane fraction, prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. Polyglutamate is then added to cover bare regions remaining on beads between attached hepatocytes. After vortexing and several washings, beads are coated with plasma membranes. Another aliquot of isolated hepatocytes served to prepare a whole cell homogenate and a microsomal fraction. The cytochrome P-450 content was measured spectrophotometrically after solubilization of proteins from membrane-coated beads by 1% lubrol PX. NADPH-cytochrome c reductase and monooxygenase activities were measured directly on membranEzcoa!ed beads. For SDS-PAGE and immunoblotting, plasma membrane proteins (30 pg/well) solubilized with the L;lemmli buffer were in&-&d with specific antibodies directed against different cytochrome P-450 isoenzymes, or with human anti-LKM, sera. Morphologic studies used enzyme-isolated, non permeabilized, fixed hepatocytes. The cells were incubated with antibodies against different isoeuzymes or with anti-LKM, human sera, and submitted to indirect immunofluorescence or immunoperoxidase methods. Results. Our plasma membrane fraction was practically devoid of microsomal contamination (less than l%), as judged from the activities of glucose6-phosphatase and NADH-cytochrome c reductase. Nevertheless, the specific content (per mg of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25% and ethoxycoumarin deethylase 11%. On immunoblots, antibodies against the constitutive rat cytochromes P-450 UT-A (IICll) and UT-N (IIDl) recognized a band comigrating with their specific antigens, in the plasma membrane fraction. Antibodies against the inducible cytochromes P-450 PB-B (IIBl), ISF-G (IA2) and PCN-E (IIlA2) also recognized one band, faint &ith untreated rat membranes and amplified with membranes from induced rats, Human sera with anti-LKM, antibodies reacted specifically with rat liver plasma membrane cytochrome P-450 UT-H (IIDl). Immunofluorescence and immunoperoxidase staining confirmed the presence of these cytochromes P-450 on the plasma membrane surface of hepatocytes. Anti-LKMi sera also stained the plasma membrane surface both on immunofluorescence and on immunoperoxidase. Conclusions. Our results show that several cytochrome P-450 isoenzymes (IICll), (IIDl), (IIBl), (IA2) and (IIIA2) are present on the plasma membrane surface of rat hepatocytes. Plasma membrane cytochrome P-450 is complete (with its protein and heme moiety), active and inducible. Conceivably, plasma membrane cytochrome P-450 may become a neoastigen in some subjects. Reference Stasiecki,P., 1980,Eur. J. Cell. Bid. 21, 79.
rences Schmidt,B., 1963,Am. J. Ophthalmol.55, 208. Okun, E., Gouras,P., 1963,Arch. Ophthalmol.69, 59. I0.tu.14.6 1
on isolated rat Loeper *, J., Desoatoire *, V., Maurice *, M., Beaune * *, P., Feldmann *, G., Larrey *, D. and Pessayre *, D. * Unit&de Recherches de Physiopathologie
HPpatique (INSERM U-24) and * * INSERM Paris, France
U-75, CHU Necker-Enfants-Malades,
Antibodies against cytochrame P-450 are found in some children with autoimmune hepatitis (anti-k& directed against cytochrome P-450 IID6) or in patients with ticrynafen hepatitis (anti-LKM,, directed against cytochrome P-450 IICIO). For such antibodies (or sensitized cytotoxic T cells) to possibly damage the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of the hepatocytes. While this presence has been reported by some authors (Stasieclciand Oesch, 1980), other authors have concluded to its absence. Aim of tb.e study. In view of the conflicting data of the literature on this important topic, we have reinvestigated the possible presence of cytochrome P-450 on the plasma membrane, by both biochemical and morphological methods. Methods. Biochemical studies were performed with a plasma membrane fraction, prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. Polyglutamate is then added to cover bare regions remaining on beads between attached hepatocytes. After vortexing and several washings, beads are coated with plasma membranes. Another aliquot of isolated hepatocytes served to prepare a whole cell homogenate and a microsomal fraction. The cytochrome P-450 content was measured spectrophotometrically after solubilization of proteins from membrane-coated beads by 1% lubrol PX. NADPH-cytochrome c reductase and monooxygenase activities were measured directly on membranEzcoa!ed beads. For SDS-PAGE and immunoblotting, plasma membrane proteins (30 pg/well) solubilized with the L;lemmli buffer were in&-&d with specific antibodies directed against different cytochrome P-450 isoenzymes, or with human anti-LKM, sera. Morphologic studies used enzyme-isolated, non permeabilized, fixed hepatocytes. The cells were incubated with antibodies against different isoeuzymes or with anti-LKM, human sera, and submitted to indirect immunofluorescence or immunoperoxidase methods. Results. Our plasma membrane fraction was practically devoid of microsomal contamination (less than l%), as judged from the activities of glucose6-phosphatase and NADH-cytochrome c reductase. Nevertheless, the specific content (per mg of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25% and ethoxycoumarin deethylase 11%. On immunoblots, antibodies against the constitutive rat cytochromes P-450 UT-A (IICll) and UT-N (IIDl) recognized a band comigrating with their specific antigens, in the plasma membrane fraction. Antibodies against the inducible cytochromes P-450 PB-B (IIBl), ISF-G (IA2) and PCN-E (IIlA2) also recognized one band, faint &ith untreated rat membranes and amplified with membranes from induced rats, Human sera with anti-LKM, antibodies reacted specifically with rat liver plasma membrane cytochrome P-450 UT-H (IIDl). Immunofluorescence and immunoperoxidase staining confirmed the presence of these cytochromes P-450 on the plasma membrane surface of hepatocytes. Anti-LKMi sera also stained the plasma membrane surface both on immunofluorescence and on immunoperoxidase. Conclusions. Our results show that several cytochrome P-450 isoenzymes (IICll), (IIDl), (IIBl), (IA2) and (IIIA2) are present on the plasma membrane surface of rat hepatocytes. Plasma membrane cytochrome P-450 is complete (with its protein and heme moiety), active and inducible. Conceivably, plasma membrane cytochrome P-450 may become a neoastigen in some subjects. Reference Stasiecki,P., 1980,Eur. J. Cell. Bid. 21, 79.