Studies on serotonin binding proteins of nerve ending membranes

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Abstract
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°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=5.3×10−7M and Kd2=1.1×10−5M. The dissociation reaction followed first order kinetics in two steps. The first step was very rapid, the second step proceeded with a half life time t1/2of 16 min and a dissociation rate constant of k−1=7.2×10−4s−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.