PARTIAL-PURIFICATION OF THE 5-HYDROXYTRYPTOPHAN-REUPTAKE SYSTEM FROM HUMAN BLOOD-PLATELETS USING A CITALOPRAM-DERIVED AFFINITY RESIN

This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and Citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50–70-fold increase in specific [3H]imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 µM Citalopram resulted in a 22% recovery of binding activity. A 10000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after 125I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of [3H]imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and [3H]imipramine binding activity. © 1990, American Chemical Society. All rights reserved.