Abstract
A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.
Highlights
From the +ClaudeBernard “Neurochimie des Communications Cellulaires,” HGpitalSaint-Louis and Facult6 de Pharmacie, Universite Paris V, the llUnit6 des Antighes Bactiriens, URA CNRS 557
A 68-kDa glycoprotein bearing thebiological activ- amine, 5-HT)’ uptake system which is present in selective ity of the plasma membrane serotonin(5-hydroxy- group of cells, such as serotoninergic neurons (Graham and tryptamine, 5-HT)transporter has been purified from Langer, 1988), blood platelets(DaPrada et al, 1988) and human blood platelets, a classical cell model for the enterochromaffin cells (Ahlman and Dahlstrom, 1982).In all study of 5-HT uptake
The purified glycoprotein bound the 5-HT uptake blocker [‘Hlparoxetine with a K d (0.25 nM) similar to theone observed for intacthuman platelets
Summary
One of the 5-HT-affinity gels, 5-HT matrix type I, already described by Sturgeon and Sturgeon (1982),was purchased from Sigma. See Ostrove and Weiss, 1990).For the 6-FTmatrix, 6-FT was coupled to 20 ml of Sepharose 4B (Pharmacia) activated by benzoquinone (Brandt et al, 1975) with diaminodipropylamine as a spacer in the presence of dicyclohexylcarbodiimide (226mg) (Tengblad, 1981), hydroxysuccinimide (140 mg), and bromoacetic acid (139 mg) at pH 8.5 (NaHC03 buffer, 0.1 M) (Cuatrecasas andParikh 1972) This mixture was incubated at room temperature for 3 days. In some instances isolated platelets were incubated 15min a t 37 "C Attempts to further improve the purification using DEAE or with 1p M paroxetine or prepared in aNa+-free Tyrode-Tris buffer. Isolated human platelets in the presence or absence of 5-HT uptake After washing (Tyrode-Tris buffer, pH 7.40, 30 ml) the wheat germ inhibitors was measured using a short incubation time (60 s) as agglutinin-agarose was eluted (90 min, 4 "C) with 10 mlof Tyrodedescribed previously (Gespach et al, 1986).The 5-HTinhibitors were Tris buffer containing 10 mM N-acetylglucosamine.
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