Leysen J E, Gommeren W
J Neurochem. 1981 Jan;36(1):201-19. doi: 10.1111/j.1471-4159.1981.tb02396.x.
I. Binding of [3H] apomorphine to dopaminergic receptors in rat striatum was most reproducible and clearly detectable when incubations were run at 25 degrees C in Tris-HCl buffer, pH 7.5, containing 1 mM-EDTA and 0.01% ascorbic acid, using a washed total-membrane fraction. The receptor binding was stereospecifically inhibited by (+)-butaclamol, and dopamine agonists and antagonists showed high binding affinity for these sites. Unlabelled apomorphine inhibited an additional nonstereospecific binding site, which was unrelated to dopamine receptors. EDTA in the incubation mixture considerably lowered nonstereospecific [3H]apomorphine binding, apparently by preventing the complexation of the catechol moiety with metal ions which were demonstrated in membrane preparations. Stereospecific [3H]apomorphine binding was not detectable in the frontal cortex, whereas in the absence of EDTA much saturable nonstereospecific binding occurred. II. Kinetic patterns of stereospecific [3H]spiperone and [3H]apomorphine binding to rat striatal membranes and the inhibition patterns of a dopamine antagonist and an agonist were evaluated at different temperatures in high-ionic-strength Tris buffer with salts added and low-ionic-strength Tris buffer with EDTA. Apparent KD values of spiperone decreased with decreasing tissue concentrations. KD values of both spiperone and apomorphine were little influenced by temperature changes. Scatchard plots of the stereospecific binding changed from linear to curved; the amount of nonstereospecific binding of the 3H ligands varied considerably, but in opposite directions for spiperone and apomorphine in the different buffers. In various assay conditions, interactions between agonists, and between antagonists, appeared fully competitive, but agonist-antagonist interactions were of mixed type. The anomalous binding patterns are interpreted in terms of surface phenomena occurring upon reactions of a ligand with complex physicochemical properties and nonsolubilized sites on membranes suspended in a buffered aqueous solution. It is concluded that anomalous binding patterns are not necessarily an indication of binding to multiple sites or involvement of distinct receptors for high-affinity agonist and antagonist binding.
I. 当使用洗涤过的全膜组分，在含有1 mM - EDTA和0.01%抗坏血酸的pH 7.5的Tris - HCl缓冲液中于25℃进行孵育时，[3H]阿扑吗啡与大鼠纹状体中多巴胺能受体的结合最为可重复且清晰可测。受体结合被(+)-布他拉莫立体特异性抑制，多巴胺激动剂和拮抗剂对这些位点表现出高结合亲和力。未标记的阿扑吗啡抑制了另一个与多巴胺受体无关的非立体特异性结合位点。孵育混合物中的EDTA显著降低了非立体特异性[3H]阿扑吗啡结合，显然是通过防止儿茶酚部分与膜制剂中证实存在的金属离子络合。在额叶皮质中未检测到立体特异性[3H]阿扑吗啡结合，而在没有EDTA的情况下，发生了大量可饱和的非立体特异性结合。II. 在添加了盐的高离子强度Tris缓冲液和含有EDTA的低离子强度Tris缓冲液中，于不同温度下评估了立体特异性[3H]螺哌隆和[3H]阿扑吗啡与大鼠纹状体膜的结合动力学模式以及多巴胺拮抗剂和激动剂的抑制模式。螺哌隆的表观KD值随组织浓度降低而降低。螺哌隆和阿扑吗啡的KD值受温度变化影响很小。立体特异性结合的Scatchard图从线性变为曲线；3H配体的非立体特异性结合量变化很大，但在不同缓冲液中螺哌隆和阿扑吗啡的变化方向相反。在各种测定条件下，激动剂之间以及拮抗剂之间的相互作用似乎完全竞争，但激动剂 - 拮抗剂相互作用为混合型。这些异常结合模式是根据具有复杂物理化学性质的配体与悬浮在缓冲水溶液中的膜上的非溶解位点反应时发生的表面现象来解释的。得出的结论是，异常结合模式不一定表明与多个位点结合或涉及高亲和力激动剂和拮抗剂结合的不同受体。
