PURIFICATION AND CHARACTERIZATION OF THE DOG HEPATIC CYTOCHROME-P-450 ISOZYME RESPONSIBLE FOR THE METABOLISM OF 2,2',4,4',5,5'-HEXACHLOROBIPHENYL
PURIFICATION AND CHARACTERIZATION OF THE DOG HEPATIC CYTOCHROME-P-450 ISOZYME RESPONSIBLE FOR THE METABOLISM OF 2,2',4,4',5,5'-HEXACHLOROBIPHENYL
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DOI：
10.1016/0003-9861(87)90396-1
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发表时间：
1987-06-01
影响因子：
3.9
通讯作者：
HALPERT, JR
中科院分区：
文献类型：
--
作者：
DUIGNAN, DB;SIPES, IG;HALPERT, JR
已经研究了 Beagle 犬和 Sprague-Dawley 大鼠的 2,2'',4,4'',5,5''-六氯联苯 (245-HCB) 肝脏代谢率显着差异的生化基础。对照狗肝微粒体代谢该底物的速度比对照大鼠肝微粒体快 15 倍。用苯巴比妥 (PB) 治疗后，狗体内至少会诱导两种细胞色素 P-450 同工酶，并且 245-HCB 的肝微粒体代谢在每纳摩尔 P-450 基础上（两倍）和每毫克蛋白质基础上（五倍）均增加。 PB 诱导的同工酶之一，PBD-2，已被纯化至 17-19 nmol/mg 蛋白质的特定含量，并且通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳证明其同质性 > 95%。在含有细胞色素 b5 的重建系统中，该同工酶显示出对 245-HCB 的活性，其活性是 PB 诱导狗的完整肝微粒体中观察到的活性的三倍以上。含有由大鼠 PB 诱导的主要同工酶 (PB-B) 的重建系统代谢 245-HCB 的速率是纯化 PBD-2 观察到的速率的 1/10。抗体抑制研究表明，在对照组和 PB 诱导的狗中，PBD-2 占 245-HCB 肝微粒体代谢的 90% 以上，而 PB-B 仅占从 PB 处理的大鼠中获得的微粒体对该化合物的代谢的约一半。免疫印迹分析显示，PB 治疗后，狗肝微粒体中的 PBD-2 水平增加了近六倍，并且这种增加与狗的 245-HCB 肝微粒体代谢率增加了五倍密切相关。这些数据共同支持了同工酶 PBD-2 在对照犬和 PB 诱导犬的 245-HCB 肝脏代谢中的主要作用。此外，这些结果表明，与大鼠相反，由于存在具有高效 245-HCB 代谢活性的细胞色素 P-450 同工酶，狗可以轻松代谢 245-HCB。
The biochemical basis for the marked difference in the rate of the hepatic metabolism of 2,2'',4,4'',5,5''-hexachlorobiphenyl (245-HCB) by Beagle dogs and Sprague-Dawley rats has been investigated. Control dog liver microsomes metabolize this substrate 15 times faster than control rat liver microsomes. Upon treatment with phenobarbital (PB), at least two cytochrome P-450 isozymes are induced in the dog, and the hepatic microsomal metabolism of 245-HCB is increased on both a per nanomole P-450 basis (twofold) and a per milligram protein basis (fivefold). One of the PB-induced isozymes, PBD-2, has been purified to a specific content of 17-19 nmol/mg protein and to > 95% homogeneity, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b5, this isozyme shows an activity toward 245-HCB which is greater than threefold that seen in intact liver microsomes from PB-induced dogs. A reconstituted system containing the major isozyme induced by PB in the rat (PB-B) metabolizes 245-HCB at 1/10 the rate observed with purified PBD-2. Antibody inhibition studies have shown that PBD-2 accounts for > 90% of the hepatic microsomal metabolism of 245-HCB in control and PB-induced dogs, while PB-B only accounts for about half of the metabolism of this compound by microsomes obtained from PB-treated rats. Immunoblot analysis has revealed that the level of PBD-2 in dog liver microsomes increases nearly sixfold with PB treatment, and this increase correlates well with the fivefold increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Together these data support a primary role for isozyme PBD-2 in the hepatic metabolism of 245-HCB in control and PB-induced dogs. In addition, these results suggest that, in contrast to rats, dogs can readily metabolize 245-HCB as a result of the presence of a cytochrome P-450 isozyme with efficient 245-HCB metabolizing activity.