Solubilization of an Adenosine Uptake Site in Brain (original) (raw)

1985, Journal of Neurochemistry

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine (13H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible. specific. and high affinity in nature. The K , and B,,, of guinea pig extracts are 0.13 t 0.02 nM and 133 c 18 fmolimg protein. respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of ['HINBI binding with the following order of potency, dilazep > hexobendine > dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine i s without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. T h e binding of t h e adenosine A , r e c e p t o r agonist [3H]cyclohexyladenosine ([3H]CHA) to the solubilized brain extract was also studied and compared with that of ['HINBI. In c o n t r a s t t o t h e [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a K , of 0.32 * 0.06 nM and a B,,, of 105-+ 30 f m o l h g protein ,and a loweraffinity site with a K , of 5.50 ? 0.52 nM and B,,, of 300 ? 5S fmol/mg protein. The pharmacology of the ['HICHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake ([3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state. Key Words: Solubilized adenosine uptake sites-Nitrobenzylthioinosine-Adenosine receptors. Verrna A. et al. Solubilization of an adenosine uptake site in brain.