北京鸭红细胞膜β肾上腺素受体放射配基结合测定法

本文以³H]dihydroalprenolol(³H]DHA)为放射配基。对北京鸭红细胞(RBC)膜β受体进行了系统研究。结果表明,³H]DHA与北京鸭RBC结合具有饱和性,肾上腺素能激动剂与³H]DHA竞争结合的强弱顺序与它们的生物活性一致,提示结合有结构特异性和立体特异性,³H]DHA与鸭RBC结合迅速、可逆。可见北京鸭RBC膜存在β受体,用于放射配基结合测定有取材方便,受体较丰富,膜蛋白得率高,³H]DHA非特异结合低和易于保存等优点,可以代替火鸡RBC。

Radioligand binding assay of a beta-adrenoceptor on duck erythrocyte membranes

(-) ³H] dihydroalprenolol (³H] DHA), a potent adrenergic antagonist, was used to identify binding sites in the erythrocyte membrane of Beijing duck. Binding of ³H] DHA to the duck erythrocyte membrane exhibited characteristics expected of β-adrenoceptor. The binding was shown to be saturable, with high affinity and K_D value of 2.95 nM and B_max value of 102 fmol/mg protein. β-Adrenoceptor agonists and antagonists compete for these binding sites in a manner directly parallels to their relative activities for β-adrenoceptor. Thus, the binding is highly stereospecific with the (-) isomer of isoprenaline being two orders of magnitude more potent than the (+) isomer in competing for these sites. The order of potency for inhibition of binding by β-adrenergic agonists was found to be (-) isoprenaline > (-) norepinephrine= (-) epinephrine> (+) isoprenaline. The binding of ³H] DHA to duck erythrocyte membrane was found to be rapid and reversible, with an association rate constant of 0.46 min^-1.nM^-1 and a dissociation rate constant of 0.42 min^-1. These results suggest that the binding sites in the erythrocyte membrane of the Beijing duck are β-adrenoccptor. In our exprements, the β-adrenoceptor of duck erythrocyte exhibited approximately the same affinity for norepinephrine and epinephrine, suggesting that β-adrenoceptor are dominant in the duck erythrocyte membrane. The density of β-adrenoceptor in the duck erythrocyte is lower than that in turkey erythrocyte. However, the membrane protein obtained per ml. duck blood may be five times as high as than that obtained per ml turkey blood. The nonspecific bindings of ³H] DHA to the duck erythrocyte membrane was shown to be very low. In addition, the preparations of the erythrocyte mebrane keeps well in deep freezrer. Since duck is more popular than turkey in China, we suggest that duck erythrocyte be used in β-adrenoceptor studies in stead of turkey erythrocyte.