Interactions among cytochromes P-450 in the endoplasmic reticulum. Detection of chemically cross-linked complexes with monoclonal antibodies.
The quaternary structure of rat liver cytochrome P-450 within microsomal membranes from 3-methyl-cholanthrene-treated rats was examined by a novel chemical cross-linking-monoclonal antibody approach. Complex formation among the different forms of P-450 was probed by cross-linking of membrane proteins followed by immunopurification with a monoclonal antibody (mAb) to P-450c, the major 3-methylcholanthrene-inducible form. Subsequent immunoblot analysis of the immunopurified proteins with this mAb indicated that P-450c formed complexes with other microsomal proteins. Immunoblots with mAbs to different P-450s were carried out to identify the P-450s that were cross-linked to P-450c. This approach detected specific cross-linking of P-450c to P-450 2a. Immunoinhibition experiments suggest that P-450 2a further metabolizes the primary phenols produced by P-450c-catalyzed hydroxylation of benzo[a]pyrene. Complex formation among membrane-bound enzymes has implications for their catalytic efficiency and an approach combining cross-linking and monoclonal antibody-based characterization of cross-linked proteins will be useful for elucidating such membrane protein macrostructures.
Alston
K
K
Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Robinson
R C
RC
Park
S S
SS
Gelboin
H V
HV
Friedman
F K
FK
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D003432:Cross-Linking Reagents; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004790:Enzyme Induction; D006900:Hydroxylation; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D003577:Cytochrome P-450 Enzyme System