[H-3] WIN 35,428 BINDING TO THE DOPAMINE UPTAKE CARRIER .1. EFFECT OFTONICITY AND BUFFER COMPOSITION

In the present study, the dopamine transporter on rat striatal membran es was labeled with [H-3]WIN 35,428 beta-carbomethoxy-3beta-(4-fluorop henyl)-tropane), 4-fluorophenyl)-tropane), and its binding state or fo rm was manipulated by changing tonicity and buffer composition. Bindin g to P2 membranes was enhanced by the presence of sucrose in the assay . This effect was not due solely to factors relating to tonicity becau se creation of isotonicity by dextrose or N-methyl-D-glucamine was les s effective, and an increase in binding by sucrose was also observed i n assays that were already isotonic by a mixture of sodium phosphate a nd NaCl. Under the latter conditions, fructose and mannose were equall y effective as sucrose. Other important factors were the presence of s odium 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 poly tronning step or in the binding assay was restricted to a decrease in the K(d) of the main binding component.