Wollemann M, Farkas J, Tóth G, Benyhe S
Institute of Biochemistry, Hungarian Academy of Sciences, Szeged.
J Neurochem. 1994 Oct;63(4):1460-5. doi: 10.1046/j.1471-4159.1994.63041460.x.
A tritiated heptapeptide, [3H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([3H]Met-enkephalin-Arg6-Phe7), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent KD value of the radioligand calculated from homologous displacement experiments was 3.4 nM, and the maximal number of specific binding sites was 630 fmol/mg of protein. The KD determined from equilibrium saturation binding studies was found to be 3.6 nM. However, the Hill coefficient was far below unity (nH = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3H]Met-enkephalin-Arg6-Phe7 binding reflects a kappa 2- and/or delta-subtype specificity of the heptapeptide. Binding to a kappa 1 and/or mu site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin-Arg6-Phe7 in frogs cannot be ruled out. The [3H]-Met-enkephalin-Arg6-Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.
为了表征其与蛙脑膜组分的阿片样物质结合活性，已合成了一种具有高比放射性的氚标记七肽，[3H]酪氨酰-甘氨酰-甘氨酰-苯丙氨酰-甲硫氨酰-精氨酰-苯丙氨酸（[3H]甲硫氨酸脑啡肽-精氨酸6-苯丙氨酸7）。从同源置换实验计算得到的放射性配体的表观解离常数（KD）值为3.4 nM，特异性结合位点的最大数量为630 fmol/mg蛋白质。从平衡饱和结合研究确定的KD为3.6 nM。然而，希尔系数远低于1（nH = 0.43），这表明存在第二个低亲和力结合位点。用左啡诺和其他一些配体进行的置换实验加强了这种结合成分的存在。假定低亲和力位点没有阿片样物质特性。所应用的配体与[3H]甲硫氨酸脑啡肽-精氨酸6-苯丙氨酸7结合进行可逆竞争时的效价顺序反映了该七肽的κ2和/或δ亚型特异性。排除了与阿片样物质受体的κ1和/或μ位点的结合，但不能排除在蛙中存在一种针对甲硫氨酸脑啡肽-精氨酸6-苯丙氨酸7的新型内源性阿片样物质受体亚型。[3H]-甲硫氨酸脑啡肽-精氨酸6-苯丙氨酸7的结合受到钠离子和GppNHp的抑制，这表明该七肽具有阿片样物质激动剂特性。
