Hanner M, Moebius F F, Weber F, Grabner M, Striessnig J, Glossmann H
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
J Biol Chem. 1995 Mar 31;270(13):7551-7. doi: 10.1074/jbc.270.13.7551.
We recently characterized (Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann H. (1993) Mol. Pharmacol. 43, 139-144) and purified (Moebius, F. F., Hanner, M., Knaus, H. G., Weber, F., Striessnig, J., and Glossmann, H. (1994) J. Biol. Chem. 269, 29314-29320) a binding protein for the phenylalkylamine Ca2+ antagonist emopamil. The emopamil-binding protein (EBP) acts as a high affinity acceptor for several antiischemic drugs and thus represents a potential common molecular target for antiischemic drug action. Degenerate oligonucleotides were synthesized according to the N-terminal amino acid sequence of purified EBP and used to amplify a guinea pig cDNA with reverse transcriptase-polymerase chain reaction and to clone full-length cDNAs from guinea pig and human liver cDNA libraries. The cDNAs coded for 229 (guinea pig) and 230 (human) amino acid 27-kDa polypeptides without significant sequence homology with any known protein. However, EBP shared structural features with pro- and eukaryotic drug transport proteins. The amino acid identity between human and guinea pig EBP was 73%. Hydrophobicity plots predicted four transmembrane segments. The C terminus contained a lysine-rich consensus sequence for the retrieval of type I integral membrane proteins to the endoplasmic reticulum. The heterologous expression of human and guinea pig EBP in Saccharomyces cerevisiae demonstrated that the expression of EBP alone is sufficient to form high affinity drug- and cation-binding domains identical to the [3H]-emopamil-binding site of guinea pig liver. Northern and Western blot analysis revealed high abundance of EBP in guinea pig epithelial tissues as liver, bowel, adrenal gland, testis, ovary, and uterus and low densities in brain, cerebellum, skeletal muscle, and heart. EBP is suggested to be the first structurally characterized member of a family of high affinity microsomal drug acceptor proteins carrying so called sigma-binding sites.
我们最近对苯烷基胺类钙离子拮抗剂埃莫帕米的一种结合蛋白进行了特性鉴定（莫比乌斯，F.F.，伯罗斯，G.G.，施特里西格，J.，和格洛斯曼，H.（1993年）《分子药理学》43卷，第139 - 144页）并进行了纯化（莫比乌斯，F.F.，汉纳，M.，克瑙斯，H.G.，韦伯，F.，施特里西格，J.，和格洛斯曼，H.（1994年）《生物化学杂志》269卷，第29314 - 29320页）。埃莫帕米结合蛋白（EBP）作为几种抗缺血药物的高亲和力受体，因此代表了抗缺血药物作用的一个潜在共同分子靶点。根据纯化后的EBP的N端氨基酸序列合成了简并寡核苷酸，并用于通过逆转录 - 聚合酶链反应扩增豚鼠cDNA，以及从豚鼠和人肝脏cDNA文库中克隆全长cDNA。这些cDNA编码了由229个（豚鼠）和230个（人）氨基酸组成的27 kDa多肽，与任何已知蛋白质均无明显序列同源性。然而，EBP与原核和真核药物转运蛋白具有共同的结构特征。人和豚鼠EBP之间的氨基酸同一性为73%。疏水性图谱预测有四个跨膜区段。C端包含一个富含赖氨酸的共有序列，用于将I型整合膜蛋白回收到内质网。人和豚鼠EBP在酿酒酵母中的异源表达表明，单独表达EBP就足以形成与豚鼠肝脏的[³H] - 埃莫帕米结合位点相同的高亲和力药物和阳离子结合结构域。Northern和Western印迹分析显示，EBP在豚鼠肝脏、肠道、肾上腺、睾丸、卵巢和子宫等上皮组织中含量丰富，而在脑、小脑、骨骼肌和心脏中含量较低。EBP被认为是携带所谓西格玛结合位点的高亲和力微粒体药物受体蛋白家族中第一个经结构鉴定的成员。
