[3H]WIN 35,428 binding to the dopamine uptake carrier. II. Effect of membrane fractionation procedure and freezing.

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 compared between fresh and frozen tissue fractionated by various protocols. Freezing striatal tissue resulted in a decrease in binding to crude or P2 membranes, but did not consistently generate upward concave Scatchard plots. Among various conditions, sodium phosphate buffers containing sucrose, employed for homogenization and for measurement of binding to freshly prepared P2 membranes, were most likely to give multiple binding component resolutions. These results allow us to reconcile the existing literature on [3H]WIN 35,428 binding and to identify (1) the 'universal' binding component common to all studies thus far, (2) a high-affinity component measured with sufficiently low ligand concentration, and (3) a low-affinity component observed with sufficiently high ligand concentration.