Solubilization of D₂ Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum

Abstract

D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of dopamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL-4B column chromatography as a large molecule, with a Stokes radius of approximately 90 A. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.

Solubilization of dopamine‐D2receptors from synaptosomal membranes of the bovine caudate nucleus

“…This detergent can also be easily removed by simple dialysis. Earlier data showed that CHAPS can be successfully used for solubilization of opiate (Simonds et al, 1980) and dopamine receptors (Lew et al, 1980;Kuno et al, 1983). This work was supported by the Chemical and Pharmaceutical Industry "Lek", Ljubljana, Yugoslavia, and partly by Scientific Research Fund of Serbia.…”

“…Dopamine receptors were isolated from several sources and solubilization was achieved with different detergents (Laduron & Ilien, 1982;Hall et al, 1983;Kuno et al, 1983). Using plant glycoside digitonin, Gorissen & Laduron (1979) reported the solubilization of binding sites from dog striatum which retained the characteristics of membranebound dopamine receptors.…”

“…There- of dopamine D2-receptors using this brain structure. Investigations of different solubilizing agents and alterations of experimental conditions showed that CHAPS, previously used for soubilization of opiate receptors (Simonds et al, 1980), brain dopamine D2-receptors (Lew et al, 1981) and recently for solubilization of dopamine D2-receptors from the bovine caudate nucleus (Kuno et al, 1983) represents the best solubilizer among several detergents from different chemical categories.…”

Dependence of critical micelle concentration of a zwitterionic detergent on ionic strength: implications in receptor solubilization

“…In addition, CHAPS has very low absorbance at 280 nm (unlike Triton X-100) and does not have circular dichroic activity in the far UV region, making it ideal for optical studies of proteins. These factors have led to extensive use of CHAPS in solubilization of membrane proteins and receptors [17,[26][27][28][29][30][31].…”

Antimuscarinic effects of antihistamines : Quantitative evaluation by receptor-binding assay.

“…Membrane preparations were prepared as described previously (6). Briefly, the cerebral cortex was homogenized in 20 volumes of ice-cold 50 mM Tris-HCI (pH 7.4) with a Kinematika Polytron PT-10 (setting 7, 20 sec).…”