Dette G A, Wesemann W
J Neural Transm. 1975;37(4):281-95. doi: 10.1007/BF01258655.
Synaptic membranes were isolated from rat brain homogenates by differential and density gradient centrifugation. Membrane proteins were solubilized by detergent buffer and assayed for serotonin-binding activity by adsorption of free 5-HT on charcoal. When the membrane extract was incubated with serotonin at +4 degrees C for various times, equilibrium was reached within 10 min. With increasing serotonin concentrations the specific part of binding was saturable whereas the non-specific part increased linear with the total 5-HT added. Kinetic analysis of the data revealed two different classes of binding sites with the apparent dissociation constants Kd1 = 5.3X10(-7) M and Kd2 = 1.1X10-5 M. The disociation reaction followed first order kinetics in two steps. The first step was very rapid, the second step proceeded with a half life time t1/2 of 16 min and a dissociation rate constant of k-1 = 7.2X10(-4) s-1. the binding was sensitive to heat and SH-blocking reagents and displaceable by serotonin in excess, d-LSD, and to a lower extent by 5-methoxytryptamine and tryptamine. The significance and localization of the binding sites at the membrane are discussed.
通过差速离心和密度梯度离心从大鼠脑匀浆中分离出突触膜。用去污剂缓冲液使膜蛋白溶解，并通过活性炭吸附游离5-羟色胺来测定血清素结合活性。当膜提取物在4℃下与血清素孵育不同时间时，10分钟内达到平衡。随着血清素浓度的增加，结合的特异性部分是可饱和的，而非特异性部分随添加的总5-羟色胺线性增加。对数据的动力学分析揭示了两类不同的结合位点，其表观解离常数Kd1 = 5.3×10(-7) M和Kd2 = 1.1×10(-5) M。解离反应分两步遵循一级动力学。第一步非常迅速，第二步的半衰期t1/2为16分钟，解离速率常数k-1 = 7.2×10(-4) s-1。结合对热和SH阻断剂敏感，可被过量的血清素、d-麦角酸二乙酰胺取代，5-甲氧基色胺和色胺在较低程度上也可取代。讨论了膜上结合位点的意义和定位。
