N-ethylmaleimide irreversibly inhibits the binding of [3H]threo-(+-)-methylphenidate to the stimulant recognition site.
N-Ethylmaleimide, a nonspecific protein modifier which reacts selectively with the sulfhydryl group of cysteinyl residues under controlled conditions, irreversibly inhibited the binding of [3H]threo-(+/-)-methylphenidate to a subset of stimulant binding sites in striatal tissue membranes from the rat in vitro. The inhibition was marked by a decrease in the Bmax of binding of the radiolabelled stimulant drug, while the KD remained unchanged. Pretreatment with excess unlabelled methylphenidate afforded complete protection from inactivation of the binding site by N-ethylmaleimide. Uptake of [3H]dopamine into striatal synaptosomes was likewise reduced after treatment with N-ethylmaleimide; pretreatment with large concentrations of methylphenidate provided partial protection from inactivation of transport. These findings suggest that the stimulant recognition site on the dopamine transport complex contains one or more cysteinyl residues.
Schweri
M M
MM
Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003342:Corpus Striatum / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D005033:Ethylmaleimide / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D008774:Methylphenidate / Q000378:metabolism* / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D014316:Tritium
D002352:Carrier Proteins; D050483:Dopamine Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D014316:Tritium; D008774:Methylphenidate; D005033:Ethylmaleimide; D004298:Dopamine