Nagy H, Goda K, Arceci R, Cianfriglia M, Mechetner E, Szabó G
Department of Biophysics and Cell Biology, University of Debrecen, Hungary.
Eur J Biochem. 2001 Apr;268(8):2416-20. doi: 10.1046/j.1432-1327.2001.02122.x.
Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.
某些单克隆抗体（mAb）亲和力的变化反映了与P-糖蛋白（Pgp）介导的药物转运相关的构象变化。在存在底物或调节剂的情况下，更多的UIC2单克隆抗体与表达Pgp的细胞结合[Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913]，而其他单克隆抗体（如MM12.10、MRK16、4E3）的结合对构象不敏感。用UIC2对Pgp+细胞进行预染色可使MM12.10单克隆抗体随后的结合减少约30-40%，这表明存在可供UIC2和MM12.10结合的Pgp分子，以及仅可供MM12.10结合的其他分子。在存在某些底物/调节剂（如长春碱、环孢素A或缬氨霉素）的情况下，与UIC2预孵育可完全消除MM12.10的反应性。然而，维拉帕米、吐温-80和硝苯地平对结合的单克隆抗体比例没有显著影响。据我们所知，这是第一个能清晰区分两类调节剂的检测方法。如对这两种现象的同时评估所示，伴随单克隆抗体竞争现象的构象变化似乎密切相关，尽管与伴随UIC2位移的构象变化并不完全相同。
