Isenberg B, Thole H, Tümmler B, Demmer A
Klinische Forschergruppe, Zentrum Biochemie and Zentrum Kinderheilkunde, Medizinische Hochschule Hannover, Germany.
Eur J Biochem. 2001 May;268(9):2629-34. doi: 10.1046/j.1432-1327.2001.02155.x.
P-glycoprotein is an ATP-dependent drug-efflux pump which can transport a diverse range of structurally and functionally unrelated substrates across the plasma membrane. Overexpression of this protein may result in multidrug resistance and is a major cause of the failure of cancer chemotherapy. The most commonly used photoreactive substrate is iodoarylazidoprazosin. Its binding domains within the P-glycoprotein have so far been inferred from indirect methods such as epitope mapping. In this study, the binding sites were refined and relocalized by direct analysis of photolabeled peptides. P-glycoprotein-containing plasma membrane vesicles of Chinese hamster ovary B30 cells were photoaffinity-labeled with iodoarylazidoprazosin. After chemical cleavage behind tryptophan residues or enzymatic cleavage behind lysine residues, the resulting 125I-labeled peptides were separated by tricine/PAGE and HPLC and subjected to Edman sequencing. The major photoaffinity binding sites of iodoarylazidoprazosin were localized in the amino-acid regions 248-312 [transmembrane segment (TM)4 to TM5], 758-800 (beyond TM7 to beyond TM8) and 1160-1218 (after the Walker A motif of the second nucleotide-binding domain). Therefore the binding pocket of iodoarylazidoprazosin is made up of at least three binding epitopes.
P-糖蛋白是一种依赖ATP的药物外排泵，它能够将多种结构和功能不相关的底物转运穿过质膜。这种蛋白质的过表达可能导致多药耐药，是癌症化疗失败的主要原因。最常用的光反应性底物是碘芳基叠氮哌唑嗪。到目前为止，其在P-糖蛋白内的结合结构域是通过间接方法（如抗原表位作图）推断出来的。在本研究中，通过对光标记肽段的直接分析，对结合位点进行了优化和重新定位。用碘芳基叠氮哌唑嗪对中国仓鼠卵巢B30细胞含P-糖蛋白的质膜囊泡进行光亲和标记。在色氨酸残基后进行化学裂解或在赖氨酸残基后进行酶解后，将得到的125I标记肽段通过三羟甲基氨基甲烷/聚丙烯酰胺凝胶电泳和高效液相色谱分离，并进行埃德曼测序。碘芳基叠氮哌唑嗪的主要光亲和结合位点位于氨基酸区域248 - 312[跨膜区段（TM）4至TM5]、758 - 800（TM7之外至TM8之外）和1160 - 1218（在第二个核苷酸结合结构域的沃克A基序之后）。因此，碘芳基叠氮哌唑嗪的结合口袋至少由三个结合表位组成。
