Partial purification of the 5-hydroxytryptamine-reuptake system from human blood platelets using a citalopram-derived affinity resin [corrected].
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 microM citalopram resulted in a 22% recovery of binding activity. A 10,000-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.
Biessen
E A
EA
Department of Physical Chemistry, University of Groningen, The Netherlands.
Horn
A S
AS
Robillard
G T
GT
D006916:5-Hydroxytryptophan / Q000302:isolation & purification*; D001792:Blood Platelets / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D015283:Citalopram; D006801:Humans; D012995:Solubility
D015283:Citalopram; D006916:5-Hydroxytryptophan