Roberts C, Bond B, White I R, Herdon H J
Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Harlow, Essex.
J Recept Signal Transduct Res. 2000 May-Aug;20(2-3):167-86. doi: 10.3109/10799890009150643.
The present study describes the solubilisation of the novel anticonvulsant, SB-204269, binding site from pig cortical membranes. Throughout the study the binding of a close analogue of this compound, [125I]-SB-217644 (trans 6- Acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b ]pyran-3R-ol) was used to monitor the success of the solubilisation procedure. [125I]-SB-217644 was an ideal mechanistic tool for quantifying the binding to this novel anticonvulsant site, with a high specific activity and affinity (K(D) of 3 nmol/l). Optimum conditions for the solubilisation of this anticonvulsant binding site were investigated using a multifactorial experimental design to assess a large number of variables. Detergent type, detergent-protein ratio, absence of Mg2+ and temperature were deemed to be important factors. However, the increases observed in binding site specific activity were minimal compared with those achieved for yields. Maximum percentage yields of binding activity (25%) were achieved with a low concentration of the zwitterionic detergent, CHAPS, in the presence of a low protein concentration. This yield was further enhanced on combining mixtures of detergents. The highest recovery (37%) was achieved with a 50:50 (v:v; 1.5 x critical micelle concentration) mixture of the ionic detergent, sodium cholate, and the non-ionic detergent, MEGA-10. In summary, we report the successful solubilisation of a novel anticonvulsant binding site, identified by its selective affinity for SB-204269 and its analogues. The recovery of nearly 40% of the target binding sites from the starting material should provide a good starting point for the purification of this protein.
本研究描述了新型抗惊厥药SB - 204269在猪皮质膜中的结合位点的增溶作用。在整个研究过程中，使用该化合物的一种紧密类似物[125I]-SB - 217644（反式6 - 乙酰基 - 4S - (3 - 碘苯甲酰氨基)-3,4 - 二氢 - 2,2 - 二甲基 - 2H - 苯并[b]吡喃 - 3R - 醇）的结合来监测增溶过程的成功与否。[125I]-SB - 217644是用于定量与这个新型抗惊厥药位点结合的理想机制工具，具有高比活性和亲和力（解离常数K(D)为3 nmol/l）。使用多因素实验设计来研究这个抗惊厥药结合位点增溶的最佳条件，以评估大量变量。去污剂类型、去污剂 - 蛋白质比例、Mg2+的缺失和温度被认为是重要因素。然而，与产量所达到的增加相比，结合位点比活性的增加观察到的最小。在低蛋白浓度存在下，使用低浓度的两性离子去污剂CHAPS可实现结合活性的最大百分比产量（25%）。在混合去污剂时，该产量进一步提高。离子去污剂胆酸钠和非离子去污剂MEGA - 10以50:50（v:v；1.5倍临界胶束浓度）的混合物可实现最高回收率（37%）。总之，我们报告了一个新型抗惊厥药结合位点的成功增溶，该位点通过其对SB - 204269及其类似物的选择性亲和力得以鉴定。从起始材料中回收近40%的目标结合位点应为该蛋白质的纯化提供一个良好的起点。
