PURIFICATION AND CHARACTERIZATION OF BOVINE CEREBRAL-CORTEX A1 ADENOSINE RECEPTOR

A1 adenosine receptors (A1AR) acting via the inhibitory guanine nucleotide binding protein inhibit adenylate cyclase activity in brain, cardiac, and adipose tissue. We now report the purification of the A1AR from bovine cerebral cortex. This A1AR is distinct from other A1ARs in that it displays an agonist potency series of N6-R-phenylisopropyladenosine (R-PIA)>N6-S-phenylisopropyladenosine > (S-PIA)>5′-N-ethylcarboxamidoadenosine (NECA) compared to the traditional potency series of R-PIA>NECA>S-PIA. The A1AR was solubilized in 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (Chaps) and then purified by chromatography on an antagonist [xanthine amine congener (XAC)]-coupled Affi-Gel 10 followed by hydroxylapatite chromatography. Following purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of Mr 36,000 by silver staining, Na125I iodination with chloramine T and photoaffinity labeling with [125I]8-[4-[[[[2-(4-aminophenyl acetylamino) ethyl] carbonyl] methyl] oxy-phenyl]-1,3-dipropylxanthine. This single protein displayed all the characteristics of the A1AR, including binding an antagonist radioligand ([3H]XAC) with high affinity (Kd = 0.7 nM) and in a saturable manner (Bmax> 4500 pmol/mg). Agonist competition curves demonstrated the expected bovine brain A1AR pharmacology: R-PIA>S-PIA>NECA. The overall yield from soluble preparation was 7%. The glycoprotein nature of the purified A1AR was determined with endo- and exoglycosidases. Deglycosylation with endoglycosidase F increased the mobility of the A1AR from Mr 36,000 to Mr, 32,000 in a single step. The A1AR was sensitive to neuraminidase but resistant to α-mannosidase, suggesting the single carbohydrate chain was of the complex type. This makes the bovine brain A1AR similar to rat brain and fat A1AR in terms of its carbohydrate chains yet the purified A1AR retains its unique agonist potency series observed in membranes. © 1990.