SOLUBILIZATION AND CHARACTERIZATION OF D-FENFLURAMINE BINDING-SITES FROM BOVINE CEREBRAL-CORTEX

Stable d-Fenfluramine binding activity was obtained in high yields, in cholate extracts of bovine cerebral cortex crude membrane preparations. Dissociation constant (K-d 17 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-[I-125]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.