Patel J, Marangos P J, Stivers J, Goodwin F K
Brain Res. 1982 Apr 8;237(1):203-14. doi: 10.1016/0006-8993(82)90568-6.
The presence of distinct binding sites for adenosine in both the CNS and PNS has been proposed in numerous studies. The recent availability of stable adenosine analogues such as cyclohexyladenosine, 2-chloroadenosine and diethylphenylxanthine has made the characterization of such a receptor feasible. In the present report the binding of N6 cyclohexyl [3H]adenosine ([3H]CHA) to rat brain synaptosomal membranes is characterized. [3H]CHA binding is saturable and exhibits a biphasic kinetic saturation profile characteristic of 2 binding sites. The high affinity site has a Kd of 0.7 nM and the low affinity site 2.4 nM. The respective Bmax values are 230 and 120 fmol/mg protein in fat forebrain. The highest density of binding sites is found in the hippocampus and subcellular distribution studies indicate that the [3H]CHA site is predominantly synaptosomal. [3H]CHA binding is highly dependent in the presence of adenosine deaminase since only 30% of the binding capacity is observed in synaptosomal membranes not treated with this enzyme. Of the many cations and anions tested only copper and zinc have effects on [3H]CHA binding. Both metals are potent inhibitors of binding with copper having an IC50 of 30 microM and zinc 150 microM. Sulfhydryl reducing and alkylating agents also inhibit binding indicating that the binding site is a sulfhydryl-dependent protein.
众多研究表明，中枢神经系统和外周神经系统中均存在腺苷的独特结合位点。近期，诸如环己基腺苷、2 -氯腺苷和二乙苯基黄嘌呤等稳定腺苷类似物的可得性，使得对这类受体进行表征成为可能。在本报告中，对N6 -环己基[³H]腺苷（[³H]CHA）与大鼠脑突触体膜的结合进行了表征。[³H]CHA结合具有饱和性，并呈现出双相动力学饱和曲线，这是两个结合位点的特征。高亲和力位点的解离常数（Kd）为0.7 nM，低亲和力位点为2.4 nM。在胖前脑中，各自的最大结合量（Bmax）值分别为230和120 fmol/mg蛋白质。结合位点密度最高的区域是海马体，亚细胞分布研究表明，[³H]CHA位点主要位于突触体。[³H]CHA结合高度依赖腺苷脱氨酶的存在，因为在未用该酶处理的突触体膜中，仅观察到30%的结合能力。在测试的众多阳离子和阴离子中，只有铜和锌对[³H]CHA结合有影响。这两种金属都是结合的有效抑制剂，铜的半数抑制浓度（IC50）为30 μM，锌为150 μM。巯基还原剂和烷基化剂也抑制结合，表明结合位点是一种依赖巯基的蛋白质。
