Ciaccio P J, Halpert J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Arch Biochem Biophys. 1989 Jun;271(2):284-99. doi: 10.1016/0003-9861(89)90279-8.
A cytochrome P450 called PBD-1 isolated from liver microsomes of an adult male Beagle dog treated with phenobarbital (PB) is structurally and functionally similar to members of the P450IIIA gene subfamily in rat and human liver microsomes. The sequence of the first 28 amino-terminal residues of PBD-1 is identical in 15 and 20 positions, respectively, to the P450IIIA forms P450p from rat and P450NF (and HLp) from human. Upon immunoblot analysis, anti-PBD-1 IgG recognizes PCNa (P450p) and PCNb (PB/PCN-E) from rat, P450NF from human, and two proteins in liver microsomes from both untreated and PB-treated dogs. Similarly, anti-PCNb IgG cross-reacts with PBD-1 and with at least one protein in microsomes from untreated dogs and two proteins in microsomes from PB-treated dogs. P450IIIA-form marker steroid 6 beta-hydroxylase activities increase 2.5-fold upon PB-treatment of dogs and are selectively inhibited by anti-PBD-1 IgG. NADPH-dependent triacetyloleandomycin (TAO) complex formation and erythromycin demethylase, also marker activities for P450IIIA forms from rats and humans, increase 4- and 5-fold in dog liver microsomes upon PB treatment, whereas immunochemically reactive PBD-1 is induced 3-fold. In microsomes from PB-treated dogs, 5 mg anti-PBD-1 IgG/nmol P450 inhibits greater than 75 and 50% of TAO complex formation and erythromycin demethylase activity, respectively. TAO complex formation is not inhibited by chloramphenicol, a selective inhibitor of the major PB-inducible dog liver cytochrome P450, PBD-2. These data suggest that PBD-1 or another immunochemically related form is responsible for a major portion of macrolide antibiotic metabolism by microsomes from PB-treated dogs and for steroid 6 beta-hydroxylation by microsomes from both untreated and PB-treated dogs. Major species differences were noted, however, in the apparent Km for 6 beta-hydroxylation of androstenedione by liver microsomes from untreated rats (24 microM), humans (380 microM), and untreated dogs (4700 microM).
从用苯巴比妥（PB）处理的成年雄性比格犬肝脏微粒体中分离出的一种细胞色素P450，称为PBD-1，其结构和功能与大鼠和人肝脏微粒体中的P450IIIA基因亚家族成员相似。PBD-1的前28个氨基末端残基序列分别在15和20个位置上与大鼠的P450IIIA形式P450p和人的P450NF（以及HLp）相同。免疫印迹分析显示，抗PBD-1 IgG可识别大鼠的PCNa（P450p）和PCNb（PB/PCN-E）、人的P450NF以及未处理和PB处理犬肝脏微粒体中的两种蛋白质。同样，抗PCNb IgG与PBD-1以及未处理犬微粒体中的至少一种蛋白质和PB处理犬微粒体中的两种蛋白质发生交叉反应。犬经PB处理后，P450IIIA形式标记类固醇6β-羟化酶活性增加2.5倍，并被抗PBD-1 IgG选择性抑制。NADPH依赖性三乙酰竹桃霉素（TAO）复合物形成和红霉素脱甲基酶（也是大鼠和人P450IIIA形式的标记活性）在犬肝脏微粒体经PB处理后分别增加4倍和5倍，而免疫化学活性PBD-1诱导增加3倍。在PB处理犬的微粒体中，5 mg抗PBD-1 IgG/nmol P450分别抑制TAO复合物形成和红霉素脱甲基酶活性的75%以上和50%。TAO复合物形成不受氯霉素抑制，氯霉素是主要的PB诱导性犬肝脏细胞色素P450即PBD-2的选择性抑制剂。这些数据表明，PBD-1或另一种免疫化学相关形式负责PB处理犬微粒体对大环内酯类抗生素代谢的主要部分，以及未处理和PB处理犬微粒体对类固醇6β-羟化作用。然而，未处理大鼠（24 microM）、人（380 microM）和未处理犬（4700 microM）肝脏微粒体对雄烯二酮6β-羟化的表观Km存在明显的物种差异。
