[H-3] WIN 35,428 BINDING TO THE DOPAMINE UPTAKE CARRIER .2. EFFECT OFMEMBRANE FRACTIONATION PROCEDURE AND FREEZING

In the present study the dopamine transporter on rat striatal membrane s was labeled with [H-3]WIN 35,428 2beta-carbomethoxy-3beta-(4-fluorop henyl)-tropane) and its binding state or form was compared between fre sh and frozen tissue fractionated by various protocols. Freezing stria tal tissue resulted in a decrease in binding to crude or P2 membranes, but did not consistently generate upward concave Scatchard plots. Amo ng various conditions, sodium phosphate buffers containing sucrose, em ployed for homogenization and for measurement of binding to freshly pr epared P2 membranes, were most likely to give multiple binding compone nt resolutions. These results allow us to reconcile the existing liter ature on [H-3]WIN 35,428 binding and to identify (1) the 'universal' b inding component common to all studies thus far, (2) a high-affinity c omponent measured with sufficiently low ligand concentration, and (3) a low-affinity component observed with sufficiently high ligand concen tration.