Solubilization of high-affinity [3H]tryptamine-binding sites from rat brain.
High-affinity binding sites for [3H]tryptamine from rat brain were solubilized by treatment with detergents. Digitonin was found to be most efficient. Triton X-100 and Chaps were less and Lubrol-PX was even less effective. The properties of [3H]tryptamine binding were essentially unchanged by solubilization with digitonin. The equilibrium dissociation constant Kd was determined as 3.7 nM; the association and dissociation rates were ka = 0.019 nM-1 min-1 and kd = 0.032 min-1, respectively, resulting in a calculated Kd of 3.6 nM. The structure activity profile of membrane-bound as well as solubilized binding sites was characterized by the high affinity of some beta-carbolines, especially harmaline, and the low affinity of 5-hydroxytryptamine. On glycerol gradient centrifugation the digitonin-[3H]tryptamine-binding-site complex sedimented at 12.8 S. Size-exclusion chromatography on Sepharose 6B revealed a Stokes' radius of 5.9 nm.