Waxman D J, Lapenson D P, Park S S, Attisano C, Gelboin H V
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Mol Pharmacol. 1987 Nov;32(5):615-24.
Cytochrome P-450 (P-450) form specificities were established for a total of nine monoclonal antibodies (MAbs) raised to four distinct rat hepatic P-450 enzymes (P-450s 2c, PB-2a, PB-4, and BNF-B), using a combination of enzyme-linked immunosorbent analysis, dot immunoblotting, Western blotting, Ouchterlony immunodiffusion, and immunoinhibition analyses. Four of the MAbs were fully (greater than or equal to 85%) inhibitory toward the corresponding immunoreactive P-450s when assayed in purified, reconstituted enzyme systems, while two of the MAbs were partially inhibitory, with a maximum of 50 or 80% inhibition achieved in the presence of saturating MAb. Inhibitory MAbs reactive with P-450s 2c, 3, and PB-4, respectively, were used to demonstrate that the formation of multiple hydroxytestosterone metabolites by each of the respective purified P-450 enzymes is reflective of their inherent catalytic specificities and not due to the presence of immunochemical distinguishable P-450 enzyme contaminants. P-450 form-specific contributions to rat hepatic microsomal steroid hormone hydroxylase activities were then assessed using the inhibitory MAbs as probes. MAb-reactive P-450 2c was shown to be the major (greater than or equal to 85%) catalyst of microsomal testosterone and androstenedione 16 alpha-hydroxylation in both untreated and beta-naphthoflavone-induced rats. However, this P-450 form catalyzed only approximately 30% of hepatic microsomal steroid 16 alpha-hydroxylase activity in phenobarbital-induced adult males, and less than or equal to 10% of steroid 16 alpha-hydroxylase activity in (phenobarbital-induced immature males or adult females, where the balance of 16 alpha-hydroxylase activity is catalyzed by MAb-reactive P-450 PB-4. Although MAb-reactive P-450 PB-4 catalyzed the majority (greater than or equal to 90%) of microsomal androstenedione 16 beta-hydroxylation in phenobarbital-induced rats, this P-450 enzyme did not contribute to the low level 16 beta-hydroxylase activity of uninduced liver samples. Finally, MAb-reactive P-450 3 catalyzed at least 85% of microsomal androstenedione 7 alpha-hydroxylation, independent of the age, sex, or induction status of the animals used as source of liver microsomes. These findings demonstrate the usefulness of MAbs as probes for the contributions of individual P-450 enzymes to the metabolism of steroid hormones susceptible to hydroxylation at multiple sites.
利用酶联免疫吸附分析、斑点免疫印迹、蛋白质免疫印迹、双向免疫扩散和免疫抑制分析等方法，确定了针对四种不同大鼠肝脏细胞色素P - 450酶（P - 450 2c、PB - 2a、PB - 4和BNF - B）产生的总共九种单克隆抗体（MAb）的细胞色素P - 450（P - 450）亚型特异性。在纯化的重组酶系统中进行测定时，其中四种MAb对相应的免疫反应性P - 450具有完全抑制作用（大于或等于85%），而另外两种MAb具有部分抑制作用，在存在饱和MAb的情况下，最大抑制率分别为50%或80%。分别与P - 450 2c、3和PB - 4反应的抑制性MAb被用于证明，各自纯化的P - 450酶形成多种羟基睾酮代谢物反映了它们固有的催化特异性，而不是由于存在免疫化学上可区分的P - 450酶污染物。然后使用抑制性MAb作为探针评估P - 450亚型对大鼠肝脏微粒体类固醇激素羟化酶活性的贡献。结果显示，在未处理的和β - 萘黄酮诱导的大鼠中，MAb反应性P - 450 2c是微粒体睾酮和雄烯二酮16α - 羟化的主要催化剂（大于或等于85%）。然而，在苯巴比妥诱导的成年雄性大鼠中，这种P - 450亚型仅催化约30%的肝脏微粒体类固醇16α - 羟化酶活性，在（苯巴比妥诱导的未成年雄性大鼠或成年雌性大鼠中，该酶催化的类固醇16α - 羟化酶活性小于或等于10%，其中16α - 羟化酶活性的其余部分由MAb反应性P - 450 PB - 4催化。尽管在苯巴比妥诱导的大鼠中，MAb反应性P - 450 PB - 4催化了大部分（大于或等于90%）微粒体雄烯二酮16β - 羟化，但这种P - 450酶对未诱导肝脏样品的低水平16β - 羟化酶活性没有贡献。最后，MAb反应性P - 4�0 3催化了至少85%的微粒体雄烯二酮7α - 羟化，与用作肝脏微粒体来源的动物的年龄、性别或诱导状态无关。这些发现证明了MAb作为探针在研究单个P - 450酶对多个位点易发生羟化的类固醇激素代谢贡献方面的有用性。
