Pergamon Life Sciences, Vol. 54, No. 15, pp. 1109-1118, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0024-32O5/94 $6.00 + .00 SOLUBILIZATION AND CHARACTERIZATION OF d-FENFLURAMINE BINDING SITES FROM BOVINE CEREBRAL CORTEX Valeria Gagliardini, Carlo Taddei, Mario Salmona, Paul Pham#, Tiziana Mennini and Maddalena Fratelli. Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea 62, 20157 Mllano, Italy and # CEA Saclay, Gif sur Yvette Cedex, France (Received in final form January 25, 1994) Summary Stable d-Fenfluramine binding activity was obtained in high yields, in cholate extracts of bovine cerebral cortex crude membrane preparations. Dissociation constant (Kd 1 7 nM), stereoselectivity and the rank order of potencies of various serotonin uptake inhibitors were similar to those measured in native membranes The inhibitory effect of Na÷ ions was also maintained in the soluble state, since the presence of 100 mM Na÷ leads to an even greater reduction of the binding than in membrane-associated binding sites. Photoaffinity labeling of soluble binding sites with p-[1251]d-Fenfluramine has led to the identification of a single specific band of molecular weight around 40-50 kDa. This suggests that d-Fenfluramine binding sites are separate molecular entities from the serotonin transporter, that belongs to a family of integral membrane proteins of 68-73 kDa molecular weight. d-Fenfluramine (d-F) is used as an anorexigenic drug in obese people. It raises extracellular 5-HT levels by inducing its release from nerve terminals and by reducing its uptake (for review see1). High-affinity [3H]d-F binding to membranes of rat brain cells has been demonstrated in vitro (2) and in vivo (3). Pharmacological studies suggest that d-F binding sites closely resemble the serotonin uptake sites, labelled by [3H]lmipramine, but with some important distinctions. One of the similarities is in their ability to bind fluoxetine, a drug that inhibits both serotonin uptake and [3H]lmipramine binding (4), and is very active as an inhibitor of [3H]d-F binding, like other serotonin uptake inhibitors. An important difference between [3H]d-F and [3H]lmipramine binding sites is, first, their response to sodium: [3H]lmipramine binding is enhanced (5), whereas [3H]d-F binding is inhibited by Na + ions in vitro (2). Differences are suggested also by the fact that midalcipram and 5-HT inhibit [3H]lmipramine binding with high and moderate affinity, respectively, whereas [3H]d-F binding is not inhibited by midalcipram, and 5-HT has very low affinity for the [3H]d-F binding sites. Moreover, while d-F has negligible affinity for the [3H]lmipramine binding sites, imipramine shows high affinity for the [3H]d-F binding sites (6).