Temperature effect on the detection of muscarinic receptor-G protein interactions in ligand binding assays.
Temperature effect on the detection of muscarinic receptor-G protein interactions in ligand binding assays.
复制标题
温度对配体结合测定中毒蕈碱受体-G 蛋白相互作用检测的影响。
DOI：
10.1016/0006-2952(88)90508-4
复制
发表时间：
1988
影响因子：
5.8
通讯作者：
Narayanan,TK
中科院分区：
文献类型：
--
作者：
Aronstam,RS;Narayanan,TK
鸟嘌呤核苷酸降低激动剂结合亲和力的能力提供了受体-G蛋白偶联的方便指示：鸟嘌呤核苷酸将毒蕈碱受体从激动剂的高亲和力状态转化为低亲和力状态。我们研究了测定温度对大鼠脑干和心房中这种偶联的影响。脑干受体的激动剂亲和力随着温度的降低而增加，反映出高亲和力构象中受体的比例更大。直接（使用[3 H] oxotremorine-M）或间接（在[3 H] N-甲基东莨菪碱/氨甲酰胆碱竞争研究中）测定的5 '-鸟苷酰亚胺二磷酸（GTP的稳定类似物）对激动剂结合的影响在16 - 20之间最大。鸟嘌呤核苷酸敏感性在0-4和37时大大降低。脑干和心房毒蕈碱受体也受到温度的影响。我们认为，高亲和力的受体-G蛋白复合物在高温下是不稳定的，从而降低激动剂的亲和力和掩蔽的鸟嘌呤核苷酸的效果。在低温下，受体-G蛋白复合物被稳定，并且在鸟嘌呤核苷酸存在下不能解离。在结合试验中监测受体-G蛋白相互作用的最佳温度为16-20 ℃。
The ability of guanine nucleotides to lower agonist binding affinity provides a convenient indication of receptor-G protein coupling: guanine nucleotides convert muscarinic receptors from high-affinity states for agonists to low-affinity states. We studied the influence of assay temperature on the demonstration of this coupling in rat brainstem and atrium. Agonist affinity of brainstem receptors increased as temperature was lowered, reflecting a greater proportion of receptors in high-affinity conformations. The influence of 5'-guanylylimidodiphosphate, a stable analog of GTP, on agonist binding, determined directly (using [3 H] oxotremorine-M) or indirectly (in [3 H] N-methylscopolamine/carbamylcholine competition studies), was greatest from 16 to 20. Guanine nucleotide sensitivity was much reduced at 0–4 and 37. Brainstem and atrial muscarinic receptors were similarly affected by temperature. We suggest that high-affinity receptor-G protein complexes are unstable at high temperatures, thereby decreasing agonist affinity and masking the guanine nucleotide effect. At low temperatures, the receptor-G protein complex is stabilized and fails to dissociate in the presence of guanine nucleotides. The optimum temperature for monitoring receptor-G protein interactions in binding assays was 16–20.