Coffey L L, Reith M E
Department of Basic Sciences, University of Illinois College of Medicine, Peoria 61656.
J Neurosci Methods. 1994 Jan;51(1):23-30. doi: 10.1016/0165-0270(94)90022-1.
In the present study, the dopamine transporter on rat striatal membranes was labeled with [3H]WIN 35,428 (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and its binding state or form was manipulated by changing tonicity and buffer composition. Binding to P2 membranes was enhanced by the presence of sucrose in the assay. This effect was not due solely to factors relating to tonicity because creation of isotonicity by dextrose or N-methyl-D-glucamine was less effective, and an increase in binding by sucrose was also observed in assays that were already isotonic by a mixture of sodium phosphate and NaCl. Under the latter conditions, fructose and mannose were equally effective as sucrose. Other important factors were the presence of sodium phosphate in the homogenizing buffer and the presence of sucrose during resuspension of the membranes. When P2 membranes were prepared from homogenates in 0.32 M sucrose, the effect of sucrose in the polytronning step or in the binding assay was restricted to a decrease in the Kd of the main binding component.
在本研究中，大鼠纹状体膜上的多巴胺转运体用[3H]WIN 35,428（2β-甲氧羰基-3β-(4-氟苯基)-托烷）进行标记，其结合状态或形式通过改变张力和缓冲液组成来调控。测定中蔗糖的存在增强了与P2膜的结合。这种效应并非仅仅归因于与张力相关的因素，因为用葡萄糖或N-甲基-D-葡糖胺产生等渗性的效果较差，并且在由磷酸钠和氯化钠的混合物已经达到等渗的测定中也观察到蔗糖使结合增加。在后一种条件下，果糖和甘露糖与蔗糖的效果相同。其他重要因素是匀浆缓冲液中磷酸钠的存在以及膜重悬过程中蔗糖的存在。当从0.32 M蔗糖匀浆中制备P2膜时，蔗糖在聚能器步骤或结合测定中的作用仅限于降低主要结合成分的解离常数（Kd）。
