High- and low-affinity [ 3H]imipramine binding sites on human platelets: separate determination and involvement of sulphur-containing bonds

Abstract

We have confirmed the presence of two different classes of [ 3H]imipramine ([ 3H]IMI) binding sites on human platelets: high-affinity (K d = 0.52 nM, B max = 1670 fmol/mg protein) and low-affinity (K d = 101 nM, B max = 8 000 fmol/mg protein) binding sites. The high-affinity component of [ 3H]IMI binding can also be obtained separately as the difference between specific [ 3H]IMI binding in Na-containing and Li-containing incubation buffer. The low-affinity component can be obtained as the difference between [ 3H]IMI binding in 50 mM Tris-HCl, 5 mM KCl, 120 mM LiCl, (pH 7.5) in the absence and presence of 0.1 mM IMI. The chemical modification of SH groups was performed with Ellman's reagent (10 mM, 40 min at 23° C). The high-affinity component of the binding was totally inhibited while the low-affinity component only decreased by 39%. No decrease in [ 3H]IMI specific binding was observed when the modification of SH groups was carried out in the presence of 1 μM IMI. The inhibition of high- and low-affinity [ 3H]IMI binding was reversible since it was completely restored by incubation of modified membranes with 1,4-dithioerythritol (DTE). The reduction of SS groups by DTE (10 mM, 1 h at 23° C) in the intact membrane preparation produced an increase in total number of binding sites of the high-affinity component of [ 3H]IMI binding by 50%.