Purification and biochemical characterization of two major cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo liver

Abstract

Two cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chick embryo liver microsomes were purified. The P-450s exhibit catalytic selectivity either for arachidonic acid metabolism, particularly epoxygenation (P-450 TCDDAA, 55 kDa), or for aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin deethylase (7-EROD) (P-450 TCDDAHH, 54.5 kDa). Turnover numbers for arachidonic acid epoxygenation, AHH, and 7-EROD, respectively, were 24.2, 0.23, and 0.45 for TCDDAA and 0.57, 9.7, and 35.5 for TCDDAHH. Both P-450s were low spin, with carbon monoxide-binding peaks at 448 nm. Their N-terminal amino acid sequences showed 80% homology and contained the sequence: PXXXSATEXL, common to CYP1A P-450s but not others. Polyclonal antibodies to TCDDAA and TCDDAHH cross-reacted with both P-450s on Western blots and immunoinhibited all TCDD-induced liver microsomal arachidonic acid metabolism, AHH, and 7-EROD. Immunoquantitation using antibodies made monospecific by immunoadsorption against the heterologous P-450 showed that TCDDAA and TCDDAHH were coinduced in liver in equal amounts and accounted for all of the TCDD-induced P-450. Enzyme assays and Western blots also showed expression of both TCDDAA and TCDDAHH in kidney but only of TCDDAHH in heart, indicating that these P-450s can be independently regulated and selectively induced. On Western blots non-immunopurified anti-TCDDAA and anti-TCDDAHH antisera recognized rat and guinea pig CYP1A1 and CYP1A2. Immunopurified TCDDAA antiserum only recognized rat CYP1A2, indicating that TCDDAA is more closely related immunochemically to CYP1A2 than to CYP1A1. The evidence that pleotropic responses to Ah receptor ligands can include induction of a P-450 that can form biologically active products from the endogenous membrane lipid, arachidonic acid, suggests that organ- and cell-specific expression of TCDD-induced P-450s could affect responses to TCDD.