Reconstitution of the human placental 5-hydroxytryptamine transporter in a catalytically active form after detergent solubilization.
The 5-hydroxytryptamine (5-HT; serotonin) transporter was solubilized from purified human placental brush border membranes by cholate in the presence of urea, and the solubilized transporter was reconstituted into proteoliposomes in a functionally active form. Solubilization of the membranes with cholate in the absence of urea inactivated the transporter. The reconstitution procedure involved precipitation of the solubilized proteins and simultaneous removal of cholate and urea by poly(ethylene glycol), and incorporation of the precipitated proteins into proteoliposomes in the presence of asolectin by a freeze-thaw/sonication technique. Optimal conditions included the use of 6% poly(ethylene glycol) during the precipitation step and an asolectin/protein ratio of 10:1 during the reconstitution step. K+ was present in the reconstitution medium. The reconstituted proteoliposomes showed the ability to transport 5-HT against a concentration gradient when an inwardly directed NaCl gradient was imposed. The 5-HT transport system in the proteoliposomes had an absolute requirement for Na+ and Cl-. The system was specific for 5-HT and was inhibited by imipramine, paroxetine and fluoxetine. The Na+/Cl-/5-HT stoichiometry was found to be 1:1:1. The transport process was electrically silent, indicating that one K+ ion was countertransported for each 5-HT molecule. The reconstituted 5-HT transporter showed high affinity for 5-HT, with an apparent Michaelis-Menten constant of 0.34 +/- 0.01 microM. It is concluded that the human placental 5-HT transporter can be solubilized and reconstituted into proteoliposomes in a transport-competent form and that the characteristics of the reconstituted transporter are similar to those of the native transporter.
Ramamoorthy
S
S
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Cool
D R
DR
Leibach
F H
FH
Mahesh
V B
VB
Ganapathy
V
V
D000838:Anions; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002412:Cations; D002458:Cell Fractionation; D019826:Cholic Acid; D002793:Cholic Acids; D005260:Female; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008871:Microvilli / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011510:Proteolipids / Q000378:metabolism; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins; D012995:Solubility; D014508:Urea
D000838:Anions; D002352:Carrier Proteins; D002412:Cations; D002793:Cholic Acids; D008081:Liposomes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011510:Proteolipids; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C020485:proteoliposomes; D012701:Serotonin; D014508:Urea; D019826:Cholic Acid