Adams D A, Edwards R J, Davies D S, Boobis A R
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Biochem Pharmacol. 1997 Jul 1;54(1):189-97. doi: 10.1016/s0006-2952(97)00167-6.
The structural similarity of related forms of P450 makes selective immunoinhibition of individual forms notoriously difficult to achieve. We report the use of a targeted antibody to overcome this problem. An antibody was raised against the synthetic peptide, Ser-Lys-Lys-Gly-Pro-Arg-Ala-Ser-Gly-Asn-Leu-Ile, corresponding to residues 291-302 of human CYP1A2. This sequence of human CYP1A2 is located in a similar position to a proinhibitory region previously identified in rat CYP1A1 and CYP1A2. The antibody bound strongly and specifically to CYP1A2 in human hepatic microsomal fraction. Binding was unaffected by denaturation of the protein. The specificity of the antibody was demonstrated by immunoblotting of human hepatic microsomal fraction where a single immunoreactive band was identified at Mr 54,000. The intensity of this band correlated strongly with high-affinity phenacetin O-deethylase activity of the microsomal fractions. In addition, the antibody bound to a single protein at Mr 54,000 in the microsomal fraction of lymphoblastoid cells expressing human CYP1A2, but not to any other recombinant P450 enzyme. CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of human hepatic microsomal fraction was inhibited >90% by whole antiserum or purified immunoglobulin. This decrease in activity represents complete inhibition of CYP1A2 activity, residual phenacetin O-deethylase activity being due to low-affinity enzymes. In contrast, the antibody, which does not bind to rat CYP1A2, had no effect on CYP1A2-dependent activity (high-affinity phenacetin O-deethylase) of rat hepatic microsomal fraction. The antiserum also had no effect on human hepatic microsomal debrisoquine 4-hydroxylase (CYP2D6) or coumarin 7-hydroxylase (CYP2A6) activities, indicating that inhibition was specific to human CYP1A2. These results demonstrate the importance of the region comprising residues 291-302 of human CYP1A2 in the catalytic activity of this enzyme.
细胞色素P450相关形式的结构相似性使得对单个形式进行选择性免疫抑制极难实现。我们报道了使用一种靶向抗体来克服这一问题。制备了一种针对合成肽Ser-Lys-Lys-Gly-Pro-Arg-Ala-Ser-Gly-Asn-Leu-Ile的抗体，该肽对应于人CYP1A2的291-302位氨基酸残基。人CYP1A2的这一序列位于与先前在大鼠CYP1A1和CYP1A2中鉴定出的促抑制区域相似的位置。该抗体与人肝微粒体组分中的CYP1A2强烈且特异性地结合。结合不受蛋白质变性的影响。通过对人肝微粒体组分进行免疫印迹证明了抗体的特异性，在Mr 54,000处鉴定出一条单一的免疫反应带。该条带的强度与微粒体组分的高亲和力非那西丁O-脱乙基酶活性密切相关。此外，该抗体与表达人CYP1A2的淋巴母细胞微粒体组分中Mr 54,000处的一种单一蛋白质结合，但不与任何其他重组P450酶结合。人肝微粒体组分的CYP1A2依赖性活性（高亲和力非那西丁O-脱乙基酶）被全抗血清或纯化的免疫球蛋白抑制>90%。活性的这种降低代表CYP1A2活性的完全抑制，残留的非那西丁O-脱乙基酶活性归因于低亲和力酶。相反，该不与大鼠CYP1A2结合的抗体对大鼠肝微粒体组分的CYP1A2依赖性活性（高亲和力非那西丁O-脱乙基酶）没有影响。该抗血清对人肝微粒体去甲异喹胍4-羟化酶（CYP2D6）或香豆素7-羟化酶（CYP2A6）活性也没有影响，表明抑制作用对人CYP1A2具有特异性。这些结果证明了人CYP1A2的291-302位氨基酸残基组成的区域在该酶催化活性中的重要性。
