Purification of D2 dopamine receptor by photoaffinity labelling, high-performance liquid chromatography and preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis.
[125I]N-azidophenethylspiperone ([125I]azido-NAPS) was used as a photoaffinity ligand for bovine D2 dopamine receptor. On photolysis, [125I]azido-NAPS was covalently incorporated into a major band of 94 kDa in bovine striatal membrane as assessed by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide gel). The labelled D2 receptor protein from striatal membrane was solubilized and subjected to HPLC using gel filtration (TSK G3000SW) and hydroxyapatite gel (Pentax SH2010C), followed by two steps of preparative SDS-PAGE. The D2 receptor protein could be obtained as a single major polypeptide on SDS-PAGE by either silver staining or autoradiography.
Usui
H
H
Department of Neuropharmacology, Niigata University, Japan.
Takahashi
Y
Y
Maeda
N
N
Mitui
H
H
Isobe
T
T
Okuyama
T
T
Nishizawa
Y
Y
Hayashi
S
S
D000345:Affinity Labels; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006882:Hydroxyapatites; D008027:Light; D011954:Receptors, Dopamine / Q000302:isolation & purification*; D017448:Receptors, Dopamine D2; D012967:Sodium Dodecyl Sulfate; D013570:Synaptic Membranes / Q000737:chemistry / Q000648:ultrastructure
D000345:Affinity Labels; D006882:Hydroxyapatites; D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; D012967:Sodium Dodecyl Sulfate