Purification and biochemical characterization of two major cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo liver.
Purification and biochemical characterization of two major cytochrome P-450 isoforms induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in chick embryo liver.
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DOI：
10.1016/s0021-9258(17)41874-6
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发表时间：
1994-02
期刊：
影响因子：
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通讯作者：
Arleen;RifkindSST;io KanetoshiSII;Jason OrlinickS;Jorge;Capdevila;Charis Lee
中科院分区：
文献类型：
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作者：
Arleen;RifkindSST;io KanetoshiSII;Jason OrlinickS;Jorge;Capdevila;Charis Lee
纯化了鸡胚肝微粒体中 2,3,7,8-四氯二苯并-对二恶英 (TCDD) 诱导的两种细胞色素 P-450 亚型。 P-450 对花生四烯酸代谢，特别是环氧化（P-450 TCDDAA，55 kDa）或芳烃羟化酶（AHH）和 7-乙氧基试卤灵脱乙基酶（7-EROD）（P-450 TCDDAHH，54.5 kDa）表现出催化选择性。 TCDDAA 的花生四烯酸环氧化、AHH 和 7-EROD 的周转数分别为 24.2、0.23 和 0.45，TCDDAHH 的周转数分别为 0.57、9.7 和 35.5。两种 P-450 均为低自旋，一氧化碳结合峰位于 448 nm。它们的 N 端氨基酸序列显示出 80% 同源性，并包含序列：PXXXSATEXL，这是 CYP1A P-450 共有的序列，但其他序列则不然。 TCDDAA 和 TCDDAHH 的多克隆抗体在蛋白质印迹上与两种 P-450 发生交叉反应，并免疫抑制所有 TCDD 诱导的肝微粒体花生四烯酸代谢、AHH 和 7-EROD。使用针对异源 P-450 的免疫吸附制成的单特异性抗体进行的免疫定量显示，TCDDAA 和 TCDDAHH 在肝脏中以等量共诱导，并且占所有 TCDD 诱导的 P-450。酶测定和蛋白质印迹还显示TCDDAA和TCDDAHH在肾脏中表达，但仅在心脏中表达TCDDAHH，这表明这些P-450可以被独立调节和选择性诱导。在蛋白质印迹上，非免疫纯化的抗 TCDDAA 和抗 TCDDAHH 抗血清可识别大鼠和豚鼠 CYP1A1 和 CYP1A2。免疫纯化的 TCDDAA 抗血清仅识别大鼠 CYP1A2，表明 TCDDAA 与 CYP1A2 的免疫化学关系比与 CYP1A1 的关系更密切。对 Ah 受体配体的多效性反应可能包括诱导 P-450 的证据，P-450 可以从内源性膜脂花生四烯酸形成生物活性产物，这表明 TCDD 诱导的 P-450 的器官和细胞特异性表达可能会影响对 TCDD 的反应。
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.