Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain.
A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a molecular mass of 35 kDa which when deglycosylated undergoes a characteristic shift in molecular mass to a sharp band at 32 kDa. In addition to the receptor, silver staining revealed polypeptides with molecular masses of 39 and 41 kDa, which are ADP-ribosylated by pertussis toxin, and 36 kDa corresponding to G protein beta-subunits.
Munshi
R
R
Department of Physiology, University of Virginia, Charlottesville 22908.
Linden
J
J
D000241:Adenosine / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives; D008970:Molecular Weight; D011983:Receptors, Purinergic / Q000302:isolation & purification*; D013806:Theophylline / Q000031:analogs & derivatives; D013873:Thionucleotides; D014970:Xanthines
D000345:Affinity Labels; D011983:Receptors, Purinergic; D013873:Thionucleotides; D014970:Xanthines; C056216:3-(3-iodo-4-amino)phenethyl-1-propyl-8-cyclopentylxanthine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C031433:8-(4-sulfophenyl)theophylline; D006160:Guanosine Triphosphate; D013806:Theophylline; D019204:GTP-Binding Proteins; D000241:Adenosine; D005033:Ethylmaleimide