FILTER-BINDING ASSAY PROCEDURE FOR THYROID-HORMONE RECEPTORS
FILTER-BINDING ASSAY PROCEDURE FOR THYROID-HORMONE RECEPTORS
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DOI：
10.1016/0003-2697(83)90280-4
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发表时间：
1983-01-01
影响因子：
2.9
通讯作者：
MORISAWA, S
中科院分区：
文献类型：
--
作者：
INOUE, A;YAMAKAWA, J;MORISAWA, S
建立了用硝酸纤维素膜滤膜测定甲状腺激素受体活性的方法。用0.4M氯化钠溶液从洗涤的大鼠肝细胞核中提取受体蛋白，与~(125)I标记的甲状腺激素(T_3[三碘甲状腺原氨酸])孵育，在2度抽吸下在纤维素酯膜上过滤。C.随后用冷缓冲液清洗过滤器，并计算125I放射性。该方法可以准确地估计受体的活性，满足活性和受体蛋白浓度之间的线性关系。当与使用Sephadex G-25柱的常规方法进行比较时，这种过滤结合方法的实用性变得显而易见。为了实际应用于常规分析，对各种过滤条件进行了考察，并建立了标准程序。用该技术测定了分离的受体的表观解离常数为1.38倍。10-10M，最适pH为T3，结合范围为8.2-8.4。
An assay procedure for thyroid hormone receptor activity which used nitrocellulose membrane filters was developed. Receptor proteins, extracted from washed rat liver nuclei with a 0.4 M NaCl solution, were incubated with 125I-labeled thyroid hormone (T3 [triiodothyronine]), and filtered on the cellullose ester membranes under suction at 2.degree. C. The filters were subsequently washed with cold buffer and counted for 125I radioactivity. The method allowed an accurate estimation of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentations. The usefulness of this filter-binding method became evident when it was compared with the conventional procedure that employs Sephadex G-25 columns. For practical application to routine assays, various filtration conditions were examined, and a standard procedure was established. Using this technique, the isolated receptors were determined to possess an apparent Kd of 1.38 .times. 10-10 M and a pH optimum of T3 binding at 8.2-8.4.