Characterization of [³H]adenosine binding to fat cell membranes

[³H]Adenosine binding to fat cell plasma membrane preparations was examined by a vacuum filtration technique. Appreciable binding of adenosine to fat cell membranes could only be demonstrated in the presence of the adenosine deaminase inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine or deoxycoformycin. The binding of adenosine to these membranes was rapid, reaching equilibrium within 10 min at 37°C, and was reversible. Bound adenosine dissociated very rapidly following a 100-fold dilution at 0°C or 22°C. Only 7% of equilibrium-bound adenosine remained at 2 min following 100-fold dilution at 22°C. Scatchard plots of equilibrium binding data were nonlinear, suggesting at least two populations of adenosine binding sites possessing different affinities for adenosine. The apparent dissociation constants, K(d), and maximum binding capacities, B(max), were 9.5 x 10⁶ M and 28 pmol/mg of protein and 9.5 x 10^-4 M and 1700 pmol/mg of protein for the high and low affinity adenosine binding sites, respectively. The high affinity sites display maximum binding at pH 7.5, above pH 7.5, or below pH 6.5 adenosine binding to the fat cell membranes declines sharply. The divalent cations calcium or magnesium, at concentrations greater than 1 mM, inhibit the binding of adenosine to the high affinity sites of the fat cell membranes. Adenosine binding is reduced by prior exposure of the membranes to trypsin, chymotrypsin, or neuraminidase or by thermal denaturation. Purine derivatives compete with adenosine for binding to the membrane site in the following potency order: adenine > ATP ≃ ADP > cyclic AMP ≃ AMP ≃ inosine. Inhibition studies of high affinity adenosine binding performed with these purine derivatives indicate this binding is to multiple components of differing specificity and not to a single, homogeneous class of binding sites. Theophylline, but not dipyridamole or p-nitrobenzylthioguanosine, is a potent inhibitor of adenosine binding to the membranes. One micromolar theophylline inhibits adenosine binding 14%. Highly purified fat cell plasma membranes possess the major portion of adenosine binding sites identified with the crude fat cell plasma membranes. The highly purified fraction likewise displayed the two populations of adenosine binding sites identified in the crude preparation. Adenosine (100 μM) did not affect the specific binding of (-)-[³H]dihydroalprenolol to fat cell plasma membranes. Neither 10 μM (-)-isoproterenol nor 10 μM(-)-alprenolol affected the ability of fat cell membranes to bind [³H]adenosine. This study demonstrates that fat cell plasma membranes possess sites which bind [³H]adenosine with high affinity. The relationship between these adenosine binding sites and the influence of adenosine on cyclic AMP levels in intact fat cells and on adenylate cyclase activity is discussed.