Studies on serotonin binding proteins of nerve ending membranes.

Synaptic membranes were isolated from rat brain homogenates by differential and density gradient centrifugation. Membrane proteins were solubilized by detergent buffer and assayed for serotonin-binding activity by adsorption of free 5-HT on charcoal. When the membrane extract was incubated with serotonin at +4 degrees C for various times, equilibrium was reached within 10 min. With increasing serotonin concentrations the specific part of binding was saturable whereas the non-specific part increased linear with the total 5-HT added. Kinetic analysis of the data revealed two different classes of binding sites with the apparent dissociation constants Kd1 = 5.3X10(-7) M and Kd2 = 1.1X10-5 M. The disociation reaction followed first order kinetics in two steps. The first step was very rapid, the second step proceeded with a half life time t1/2 of 16 min and a dissociation rate constant of k-1 = 7.2X10(-4) s-1. the binding was sensitive to heat and SH-blocking reagents and displaceable by serotonin in excess, d-LSD, and to a lower extent by 5-methoxytryptamine and tryptamine. The significance and localization of the binding sites at the membrane are discussed.