Leynadier D, Peyrot V, Sarrazin M, Briand C, Andreu J M, Rener G A, Temple C
Groupe de Recherche sur les Interactions des Proteines en Pharmacologie, Faculté de Pharmacie, Marseille, France.
Biochemistry. 1993 Oct 12;32(40):10675-82. doi: 10.1021/bi00091a018.
Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 +/- 0.5) x 10(6) M-1 and (4.1 +/- 0.9) x 10(6) M-1 for the R- and S-isomer, respectively, and by several low-affinity sites. Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.
对两种对映体NSC 613862（S）-（-）和NSC 613863（R）-（+）的几种荧光特性进行了比较。尽管这两种异构体在不同溶剂的溶液中表现出相同的荧光行为，但在与纯化的小牛脑微管蛋白结合后观察到了显著差异。通过荧光光谱法和柱凝胶渗透法（一种直接测量方法）对这两种化合物进行了结合测量。对于两种异构体，结合的特征是存在一个高亲和力结合位点，R-异构体和S-异构体的表观缔合常数分别为（3.2±0.5）×10⁶ M⁻¹和（4.1±0.9）×10⁶ M⁻¹，以及几个低亲和力位点。两种异构体还均显示出能诱导微管蛋白中的GTP酶活性。这两种异构体的高亲和力结合位点似乎相同。此外，荧光竞争实验表明秋水仙碱和鬼臼毒素位点至少部分重叠。为了解释与微管蛋白结合后荧光行为的差异，我们假设R-异构体与其结合位点的位置与S-异构体不同。
