Characterization of [125I]RTI-55 binding to dog caudate dopamine transporter.
We have characterized binding of [125I]RTI-55 ((-)-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane) to the dog caudate membrane and detergent solubilized dopamine transporter sites. The solubilized transporter was assayed by filtration over polyethylenimine treated Whatman GF/B filters. [125I]RTI-55 specifically binds to transporter sites in membranes and solubilized protein with Kd of 0.27 and 0.34 nM, respectively. The binding of [125I]RTI-55 to the membrane and solubilized transporter was inhibited by dopamine, norepinephrine and serotonin uptake inhibitors with rank order and potencies consistent with that of the dopamine transporter. Thus, similar pharmacological properties between membranes and solubilized dopamine transporters were retained upon solubilization. Rapid assay of solubilized protein will aid in monitoring transporter protein purification.
Patel
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Neuroscience Branch, National Institute of Health, National Institute on Drug Abuse, Baltimore, MD 21224.
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002421:Caudate Nucleus / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002793:Cholic Acids; D015283:Citalopram / Q000494:pharmacology; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D003891:Desipramine / Q000494:pharmacology; D004285:Dogs; D050483:Dopamine Plasma Membrane Transport Proteins; D005374:Filtration; D007457:Iodine Radioisotopes / Q000378:metabolism; D008454:Mazindol / Q000494:pharmacology; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D011485:Protein Binding / Q000187:drug effects; D012995:Solubility
D002352:Carrier Proteins; D002793:Cholic Acids; D050483:Dopamine Plasma Membrane Transport Proteins; D007457:Iodine Radioisotopes; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D015283:Citalopram; C069723:2beta-carbomethoxy-3beta-(4-iodophenyl)tropane; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D008454:Mazindol; D003042:Cocaine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D003891:Desipramine