Purification and immunological characterization of a novel cytochrome P450 from C3H/10T1/2 cells.
The major polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 in the mouse embryo fibroblast-derived C3H/10T1/2 CL8 cell line (P450-EF) has been partially purified from benz[a]anthracene (BA)-induced 10T1/2 cells (40 pmol P450/mg). The purification of P450-EF was carried out by sequential chromatography of solubilized microsomes over hydrophobic aminohexyl-Sepharose 4B, anion exchange DE-52 cellulose, and cation exchange carboxymethyl trisacryl columns. The final preparation (1700 pmol/mg) appeared as a single major 55-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reconstitution of detergent-free partially purified P450-EF yielded a relatively high turnover for 7,12-dimethylbenz[a]anthracene (DMBA) metabolism (5.4 nmol/nmol/min). Polyclonal antibodies to purified P450-EF (anti-P450-EF), raised in, respectively, rabbit and chicken, detected a single 55-kDa band in 10T1/2 cell microsomes that was highly inducible by BA (approximately 20-fold) and TCDD (approximately 5-fold). Rabbit anti-P450-EF was much more effective than the corresponding chicken antibody at binding denatured P450-EF protein on Western blots. Conversely, only the chicken antibody was effective at inhibiting DMBA metabolism catalyzed by microsomal P450-EF. This antibody did not inhibit P450IA1-mediated DMBA metabolism. Rabbit anti-P450-EF recognized very weakly (less than 1% of homologous protein response) pure P450IA1, IIB1, IIC7, IIE1, and IIIA1 proteins on Western blots but exhibited substantial cross-reactivity (approximately 10%) with pure P450IIA1 and very strong cross-reactivity (approximately 75%) with a hormonally regulated rat adrenal P450. Polyclonal antibodies to several major P450 subfamilies either did not recognize P450-EF (anti-P450IA, IIB, and IIC) or recognized it very weakly (anti-P450IIA1). P450-EF is probably distantly related to the P450IIA subfamily and may belong to a new P450 subfamily.
Pottenger
L H
LH
Department of Pharmacology, University of Wisconsin, Madison 53706.
Christou
M
M
Jefcoate
C R
CR
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D001551:Benz(a)Anthracenes / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008809:Mice, Inbred C3H; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight
D000906:Antibodies; D001551:Benz(a)Anthracenes; D007527:Isoenzymes; D015127:9,10-Dimethyl-1,2-benzanthracene; D003577:Cytochrome P-450 Enzyme System; C030935:benz(a)anthracene