Properties of solubilized and reconstituted A1 adenosine receptors from bovine brain.
A simple method for solubilization and reconstitution of the A1 adenosine receptor from bovine brain is presented. Solubilization with CHAPS-phosphatidylcholine (CHAPS/PC) mixture did not alter the binding properties of the A1 adenosine receptor antagonist [3H]-DPCPX. The solubilized receptors were chromatographed on hydroxyapatite or DEAE-cellulose to remove native membrane lipids and part of non-receptor proteins. Elution of the receptor fractions was obtained from DEAE-cellulose column with a linear gradient of KCl (0-0.4 M). The fractions corresponding to the peak of [3H]-DPCPX binding activity were then reconstituted in phosphatidylcholine by dialysis. The reconstituted receptor retained all the binding characteristics and the same rank order of competition potency (R-PIA greater than S-PIA greater than NECA) as the native receptor, although its thermal stability was remarkably reduced. The binding of [3H]-DPCPX to A1 adenosine receptors was increased by GTP, probably as result of interactions with coeluted G-proteins.
Ragazzi
E
E
Department of Medicine, College of Medicine, University of Florida, Gainesville 32610.
Shryock
J
J
Palczewski
K
K
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D001667:Binding, Competitive; D001923:Brain Chemistry; D002417:Cattle; D002793:Cholic Acids; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006882:Hydroxyapatites; D066298:In Vitro Techniques; D008566:Membranes / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry; D010660:Phenylisopropyladenosine / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000737:chemistry; D011983:Receptors, Purinergic / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014970:Xanthines / Q000494:pharmacology
D002793:Cholic Acids; D006882:Hydroxyapatites; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D011983:Receptors, Purinergic; D014970:Xanthines; D010660:Phenylisopropyladenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); D006160:Guanosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019204:GTP-Binding Proteins; D000241:Adenosine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate