Solubilization of stable adenosine A1 receptors from rat brain.
Despite numerous reports of solubilization of adenosine A1 receptors, little progress has been made in isolating or purifying the receptor, owing to the extreme lability of the preparations. The present solubilization strategies recognized the possible role of endogenous adenosine to produce adenosine-receptor-N-protein complexes, which are intrinsically unstable, and instead attempted to use caffeine to solubilize free adenosine receptors, which might be more stable. Endogenous adenosine was removed from membranes by using adenosine deaminase along with GTP to accelerate the release of receptor-bound adenosine. The receptors were then occupied with caffeine and solubilized with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) in the presence of glycerol. These soluble preparations exhibited the characteristics of free adenosine receptors. They bound the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPDPX) with high affinity to a single class of binding sites, which were insensitive to GTP. The binding activity was extremely stable, with a half-life of about 5 days at 4 degrees C; there was little change in either receptor number or affinity during 3 days at 4 degrees C. This methodology should greatly facilitate the characterization, isolation and purification of the adenosine A1 receptor.
Helmke
S M
SM
Department of Pharmacology, University of Colorado Health Sciences Center, Denver 80262.
Cooper
D M
DM
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane; D002793:Cholic Acids / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011983:Receptors, Purinergic / Q000378:metabolism*; D012995:Solubility; D014970:Xanthines / Q000378:metabolism
D002793:Cholic Acids; D011983:Receptors, Purinergic; D014970:Xanthines; D006160:Guanosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate