[3H]WIN 35,428 binding to the dopamine uptake carrier. I. Effect of tonicity and buffer composition.
In the present study, the dopamine transporter on rat striatal membranes was labeled with [3H]WIN 35,428 (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and its binding state or form was manipulated by changing tonicity and buffer composition. Binding to P2 membranes was enhanced by the presence of sucrose in the assay. This effect was not due solely to factors relating to tonicity because creation of isotonicity by dextrose or N-methyl-D-glucamine was less effective, and an increase in binding by sucrose was also observed in assays that were already isotonic by a mixture of sodium phosphate and NaCl. Under the latter conditions, fructose and mannose were equally effective as sucrose. Other important factors were the presence of sodium phosphate in the homogenizing buffer and the presence of sucrose during resuspension of the membranes. When P2 membranes were prepared from homogenates in 0.32 M sucrose, the effect of sucrose in the polytronning step or in the binding assay was restricted to a decrease in the Kd of the main binding component.
Coffey
L L
LL
Department of Basic Sciences, University of Illinois College of Medicine, Peoria 61656.
Reith
M E
ME
D000818:Animals; D002021:Buffers; D002352:Carrier Proteins / Q000378:metabolism*; D003042:Cocaine / Q000031:analogs & derivatives* / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D005260:Female; D007038:Hypotonic Solutions / Q000494:pharmacology; D007552:Isotonic Solutions / Q000494:pharmacology; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010710:Phosphates / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000494:pharmacology; D013395:Sucrose / Q000378:metabolism; D014316:Tritium
D002021:Buffers; D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D010710:Phosphates; D014316:Tritium; C021279:(1R-(exo,exo))-3-(4-fluorophenyl)-8-methyl-8- azabicyclo(3.2.1)octane-2-carboxylic acid, methyl ester; D013395:Sucrose; D012964:Sodium; D003042:Cocaine; C018279:sodium phosphate; D004298:Dopamine