绍鸭垂体LHRH放射受体结合法及其结合特性

A RADIORECEPTOR ASSAY OF LUTEINIZING HORMONE RELEASING HORMONE RECEPTOR AND CHARACTERIZATION OF LHRH BINDING TO PITUITARY RECEPTORS IN SHAO DUCK

以促性腺激素释放激素(LHRH) 的高效类似物(DLys6)LHRH 作为示踪物。在垂体膜制剂和胶原酶及胰酶分散的体外培养的垂体细胞水平上,研究了绍鸭垂体LHRH 受体的结合特性。106 个垂体细胞与125I( DLys6) LHRH 在4 ℃孵育90 min能获得较高的结合率,经24h 孵育后结合率显著下降。125I(DLys6)LHRH 与垂体膜制剂的结合率随膜制剂的浓度增加而增加,在每管相当于1 ～2 个垂体浓度范围内呈正相关。特异性结合随125 I( DLys6 ) LHRH 量的增加而增加, 实验结果经Scatchard 分析揭示出特异性结合与结合和游离125I( DLys6)LHRH 之比呈线性关系,预示绍鸭垂体对( DLys6)LHRH 存在同一类高亲和力的结合位点。垂体膜制剂和垂体整体细胞的平衡解离常数( Kd) 分别为0-34n M 和0-43n M ,两个Kd 值相近且垂体整体细胞显示出较大的最大结合容量(Bmax) ,这说明本研究的酶法消化分散垂体细胞对其LHRH 受体的损伤较小。( DLys6) LHRH(10 - 11 ～10 - 6 M) 能取代示踪物的特异性结合。本研究结果验证了绍鸭垂体LHRH 受体的存在并提供了其基本结合特性。

The properties of Shao duck pituitary luteinizing hormone releasing hormone (LHRH) receptors were analyzed in pituitary membrane preparation and isolated pituitary cells prepared by enzymatic dispersion with collagenase and trypsin, by using a superagonist analog of (D Lys 6)LHRH. High binding of 125 I (D Lys 6)LHRH to 10 6 cultured cells of Shao duck was observed after a 90 minute incubation at 4℃, while binding was significantly reduced after a 24h incubation. Binding of the radioligand was a function of tissue concentration of Shao duck pituitary membrane preparation, with a positive correlation over the range of 1～2 pituitary per tube. Specific binding for 125 I (D Lys 6)LHRH increased with the increase in the amount of 125 I (D Lys 6)LHRH. The Scatchard analysis of data revealed a linear relationship between the amount of specific binding and the ratio of specific binding to free 125 I (D Lys 6)LHRH, indicating a single class of high affinity sites. Equilibrium dissociation constant(Kd) was 0 34 nM in pituitary membrane preparation and 0 43nM in isolated pituitary cells. Both Kd values were near and the maximum binding capacity (B max )was great in isolated cells, suggesting no significant loss of the LHRH receptor population caused by the enzymatic procedure employed for cell dispersion in the present study. Addition of (D Lys 6)LHRH displaced bound 125 I (D Lys 6)LHRH. These results demonstrated the presence and provided characterization of LHRH receptors in Shao duck pituitary.