Mazzoni M R, Lucacchini A
Istituto Policattedra di Discipline Biologiche, University of Pisa, Italy.
Biotechnol Appl Biochem. 1987 Aug;9(4):339-45.
Using bovine vesicular gland microsomes and [14C]indomethacin we demonstrated the presence of a specific binding site for nonsteroidal anti-inflammatory drugs. Specific binding of [14C]indomethacin to microsomes was rapid, with most of the ligand bound by 2 min at 4 degrees C. In routine binding assays the incubation temperature was maintained at 4 degrees C, because the maximal specific binding was obtained. Specific [14C]indomethacin binding appeared to increase linearly with increasing protein concentration over the range of 0.1-1.0 mg of microsomal protein. Specific binding was saturable and Scatchard analysis of binding data showed a single class of binding sites with a dissociation constant (Kd) of 3.8 microM and a maximal number of binding sites (Bmax) of about 1272 pmol/mg of protein. When these binding data were plotted according to the Hill equation, a straight line was obtained with a Hill coefficient of 1.0. Structural specificity of the nonsteroidal anti-inflammatory drug site was studied with diclofenac, arylpropionic acids (ketoprofen and indoprofen), and aspirin. Diclofenac and arylpropionic derivatives were able to compete with [14C]indomethacin for binding to microsomes, while aspirin was a weak inhibitor.
我们使用牛精囊微粒体和[14C]吲哚美辛证明了非甾体抗炎药存在特异性结合位点。[14C]吲哚美辛与微粒体的特异性结合很快，在4℃下2分钟内大部分配体就已结合。在常规结合试验中，孵育温度维持在4℃，因为此时可获得最大特异性结合。在0.1 - 1.0毫克微粒体蛋白范围内，特异性[14C]吲哚美辛结合似乎随蛋白浓度增加呈线性增加。特异性结合是可饱和的，结合数据的Scatchard分析显示存在一类结合位点，解离常数（Kd）为3.8微摩尔，最大结合位点数（Bmax）约为1272皮摩尔/毫克蛋白。当根据Hill方程绘制这些结合数据时，得到一条直线，Hill系数为1.0。我们用双氯芬酸、芳基丙酸（酮洛芬和吲哚洛芬）和阿司匹林研究了非甾体抗炎药位点的结构特异性。双氯芬酸和芳基丙酸衍生物能够与[14C]吲哚美辛竞争结合微粒体，而阿司匹林是一种弱抑制剂。
