ROLES OF SULFHYDRYL AND DISULFIDE GROUPS IN THE BINDING OF CP-55,940 TO RAT-BRAIN CANNABINOID RECEPTOR

The roles of sulfhydryl and disulfide groups in the specific binding o f synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membr ane preparations from the rat cerebral cortex have been examined. Vari ous sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and me thyl methanethiosulfonate (MMTS) inhibited the specific binding of [H- 3]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. Ab out 80-95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding af finity (K(d)) and capacity (B(max)). On the other hand, DL-dithiothrei tol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [H-3]CP-55,940 to the receptor and about 50% inhi bition was obtained at a 5 mM concentration. Furthermore, 5 mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [H-3]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essentia l to the binding of the ligand to the receptor. In addition, the inhib ition of the binding by DTT implies that besides free sulfhydryl group (s), the integrity of a disulfide bridge is also important for [H-3]CP -55,940 binding to the cannabinoid receptor.