Laurenzana E M, Sorrels S L, Owens S M
Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Drug Metab Dispos. 1995 Feb;23(2):271-8.
Four peptides (pep23-33, pep26-38, pep283-297, and pep409-419) corresponding to unique sequences in rat cytochrome P450 (CYP) 2D1 and/or human CYP2D6 were selected for production of antipeptide antibodies. Rat liver microsomal protein was recognized by anti-serum to all four peptides in ELISAs; however, antisera against pep23-33 and pep26-36 proved not usable for any other applications. Western blots of microsomal protein from a cell line specifically expressing human CYP2D6 revealed that antisera to pep283-297 and pep409-419 recognized a single protein band of the same molecular size as CYP2D6. Antisera to pep283-297 and pep409-419 recognized a rat microsomal protein presumed to be CYP2D1, because it comigrated with human CYP2D6 and had an apparent molecular size of 52 kDa. An unknown protein of approximately 85 kDa was also recognized by pep409-419. Recognition of microsomal protein(s) by antisera to pep283-297 and pep409-419 was blocked by pep283-297 or a bovine serum albumin-pep409-419 conjugate, respectively. Antiserum to pep283-297 was used to analyze sex and strain differences in liver microsomes prepared from Sprague-Dawley, Fischer 344, and Dark Agouti male and female rats. Sprague-Dawley and Fischer 344 rats expressed similar amounts of CYP2D1, but expression in Dark Agouti rats was significantly lower. The antiserum did not detect a sexual dimorphism in any of the strains. A significant correlation between antipeptide283-297 immunoreactivity and Vmax for dextromethorphan O-demethylation existed in female rat strains; however, this relationship did not exist in male rat strains.(ABSTRACT TRUNCATED AT 250 WORDS)
选择了与大鼠细胞色素P450（CYP）2D1和/或人CYP2D6中独特序列相对应的四种肽（pep23 - 33、pep26 - 38、pep283 - 297和pep409 - 419）来制备抗肽抗体。在酶联免疫吸附测定（ELISA）中，大鼠肝微粒体蛋白能被针对所有四种肽的抗血清识别；然而，针对pep23 - 33和pep26 - 36的抗血清被证明不适用于任何其他用途。对一个特异性表达人CYP2D6的细胞系的微粒体蛋白进行的蛋白质印迹分析显示，针对pep283 - 297和pep409 - 419的抗血清识别出一条与CYP2D6分子大小相同的单一蛋白条带。针对pep283 - 297和pep409 - 419的抗血清识别出一种推测为CYP2D1的大鼠微粒体蛋白，因为它与人类CYP2D6迁移率相同，表观分子大小为52 kDa。一种约85 kDa的未知蛋白也能被pep409 - 419识别。针对pep283 - 297和pep409 - 419的抗血清对微粒体蛋白的识别分别被pep283 - 297或牛血清白蛋白 - pep409 - 419偶联物阻断。针对pep283 - 297的抗血清用于分析从Sprague - Dawley、Fischer 344和Dark Agouti雄性和雌性大鼠制备的肝微粒体中的性别和品系差异。Sprague - Dawley和Fischer 344大鼠表达的CYP2D1量相似，但Dark Agouti大鼠中的表达明显较低。该抗血清在任何品系中均未检测到性别差异。在雌性大鼠品系中，抗肽283 - 297免疫反应性与右美沙芬O - 去甲基化的Vmax之间存在显著相关性；然而，在雄性大鼠品系中不存在这种关系。（摘要截短于250字）
