A novel affinity purification of D-1 dopamine receptors from rat striatum.
When rat striatal membranes were pretreated with the sulfhydryl (-SH) modifying reagent, N-ethylmaleimide (NEM) in the presence of the D-1-specific agonist, SKF R-38393, the D-1 dopamine receptor was completely protected from NEM-mediated inactivation. The D-1 receptors, solubilized from these membranes with 1% sodium cholate in the presence of phospholipids, bound with high efficiency (greater than 90%) to mercury-agarose columns. The bound receptors were eluted from the affinity column with a -SH reducing agent, beta-mercaptoethanol. Upon removal of beta-mercaptoethanol from the eluted fractions by inclusion chromatography, the receptor was reconstituted into phospholipid vesicles and assayed for ligand binding activity. The affinity purified receptor exhibited saturable and specific binding of the D-1-specific ligand 125I-SCH 23982, with a Kd of 1.6 nM comparable to that measured in intact membranes and solubilized extracts. The binding capacity of these receptors for 125I-SCH 23982 was 11,000 pmol/mg protein, representing greater than an 8000-fold purification over the starting membrane preparation. The purity of the affinity eluted receptors was estimated to be 78%. The purified receptors retained the pharmacological properties of membrane-bound receptors, including the ability to distinguish between active and inactive enantiomers of specific dopaminergic antagonists. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining revealed the presence of two major polypeptides of 74 and 54 kDa. These two polypeptides were absent in those affinity eluted fractions which did not display 125I-SCH 23982-binding activity and also were not detected in preparations obtained from membranes which were NEM-treated in the absence of D-1-specific agonist. The molecular weights of these polypeptides were similar to those of membrane-bound D-1 receptors, when labeled with a D-1-specific photo-affinity ligand, 125I-8-hydroxy-3-methyl-1-(4-azidphenyl)-2,3,4,5-tetrahydro-1H-3-b enzazepine. These two polypeptides may represent glycosylated and deglycosylated forms of the D-1 dopamine receptor.
Sidhu
A
A
Membrane Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
D000818:Animals; D001552:Benzazepines / Q000031:analogs & derivatives* / Q000378:metabolism; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003342:Corpus Striatum / Q000378:metabolism*; D018492:Dopamine Antagonists; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D011869:Radioligand Assay; D051381:Rats; D011954:Receptors, Dopamine / Q000302:isolation & purification* / Q000378:metabolism; D017447:Receptors, Dopamine D1; D012995:Solubility
C049558:8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol; D001552:Benzazepines; D018492:Dopamine Antagonists; D011954:Receptors, Dopamine; D017447:Receptors, Dopamine D1; D005033:Ethylmaleimide