Moebius F F, Hanner M, Knaus H G, Weber F, Striessnig J, Glossmann H
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
J Biol Chem. 1994 Nov 18;269(46):29314-20.
A high affinity phenylalkylamine Ca2+ antagonist binding polypeptide (Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann, H. (1993) Mol. Pharmacol. 43, 139-148) was purified to homogeneity from the endoplasmic reticulum of guinea pig liver with the aid of [3H]emopamil, an antiischemic agent, and [3H]azidopamil, a photoaffinity label. The purified protein retained its high affinity for the antiischemic drugs emopamil (Kd = 4 nM), opipramol (IC50 = 15 nM), trifluoperazine (IC50 = 2 nM), and for Zn2+ (IC50 = 2 microM). Ferguson plots revealed a molecular mass of 27.2 kDa. Partial amino acid sequence information was obtained by Edman degradation and revealed no homology to known protein sequences. Antibodies raised against a synthetic peptide corresponding to the first 25 NH2-terminal amino acid residues specifically immunoprecipitated the [3H]azidopamil photoaffinity-labeled polypeptide and recognized the protein in Western blots. Cross-linking with a variety of homo- and heterobifunctional agents lead to the formation of dimers. Since in the purified preparation no other subunit could be identified with different protein stains, our results indicate that the [3H]emopamil binding site is formed by the homodimer of a novel membrane protein.
借助抗缺血药物[3H]依莫帕明和光亲和标记物[3H]叠氮帕米，从豚鼠肝脏内质网中纯化出一种高亲和力的苯烷基胺Ca2+拮抗剂结合多肽（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，139 - 148页），使其达到同质状态。纯化后的蛋白质对抗缺血药物依莫帕明（解离常数Kd = 4 nM）、奥匹哌醇（半数抑制浓度IC50 = 15 nM）、三氟拉嗪（半数抑制浓度IC50 = 2 nM）以及对Zn2+（半数抑制浓度IC50 = 2 microM）仍保持高亲和力。弗格森图显示其分子量为27.2 kDa。通过埃德曼降解获得了部分氨基酸序列信息，结果表明该序列与已知蛋白质序列无同源性。针对对应前25个氨基末端氨基酸残基的合成肽产生的抗体，特异性免疫沉淀了[3H]叠氮帕米光亲和标记的多肽，并在蛋白质免疫印迹中识别出该蛋白质。与多种同双功能和异双功能试剂交联导致形成二聚体。由于在纯化制剂中用不同蛋白质染色法未鉴定出其他亚基，我们的结果表明[3H]依莫帕明结合位点由一种新型膜蛋白的同二聚体形成。
