Partial purification and characterization of the human placental serotonin transporter.
The human placental serotonin transporter was solubilized from purified brush border membranes using digitonin as the solubilizing agent. The solubilizate was subjected to wheat germ agglutinin-Sepharose 6B column chromatography, Centricon-100 ultrafiltration and Sepharose 6B gel filtration to yield a partially purified preparation of the serotonin transporter. Specific binding of the high affinity ligand paroxetine was used to monitor the transporter during the solubilization and the purification steps. The enrichment of paroxetine binding in the final preparation was 51-fold compared to the intact brush border membranes, taking into account the inactivation that occurred during purification. The partially purified transporter exhibited paroxetine binding characteristics which were similar to those of the transporter in intact membranes. The transporter in the partially purified preparation bound paroxetine with a high affinity (dissociation constant, 0.21 nM). The binding was inhibitable by serotonin but not by other monoamines, dopamine and norepinephrine, nor by the serotonin precursor 5-hydroxytryptophan. The antidepressants, imipramine, fluoxetine and desipramine inhibited the binding with a rank order of potency of imipramine = fluoxetine > desipramine. The approximate molecular weight of the transporter was assessed by molecular sieve chromatography on Sepharose 6B and was found to be 300,000. When reconstituted into proteoliposomes, the partially purified transporter was able to catalyse NaCl-dependent serotonin transport in these proteoliposomes. The results of this study show that the human placental serotonin transporter can be solubilized, partially purified and reconstituted in a transport-competent form and, in addition, provide some insight into the protein nature of the transporter.
Ramamoorthy
S
S
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Leibach
F H
FH
Mahesh
V B
VB
Ganapathy
V
V
D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002824:Chorionic Villi / Q000737:chemistry*; D002850:Chromatography, Gel; D003902:Detergents; D004072:Digitonin; D005260:Female; D006801:Humans; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D017374:Paroxetine / Q000378:metabolism; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D011510:Proteolipids; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D012995:Solubility
D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011257:Pregnancy Proteins; D011510:Proteolipids; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C020485:proteoliposomes; D012701:Serotonin; D017374:Paroxetine; D004072:Digitonin