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我们用(一)~3H-双氢心得舒(简称~3H-DHA、33Ci/mmol)为放射性配基,测定了β-肾上腺能受体的结合容量(R)_T,亲和力Kn(1/K_D)和Hill系数H_n;用竞争反应测定了非放射性配基(激动剂和拮抗剂)的表观解离常数K_I,观察了甲状腺激素(T_3)对大鼠脑组织β受体的调节作用。将约300μg的膜蛋白与不同浓度的~3H-DHA混合,非特异结合管另加消旋心得安(最终浓度10~(-4)M),反应总体积25μl。30℃保温15分钟,反应终了立即用国产玻璃纤维膜抽滤,用20ml按1:3稀释的冷缓冲液洗,分离结合和游离的~3H-DHA。滤膜干燥后置二甲苯闪烁液内,
We measured the binding capacity of β-adrenergic receptor (R) _T, the affinity of Kn (1 / K_D) using radioimmunoassay with 3H-dihydrochlorothiazide (~ 3H-DHA, 33Ci / mmol) ) And Hill coefficient H_n. The apparent dissociation constants K_I of non-radioactive ligands (agonists and antagonists) were determined by competitive reaction. The regulatory effect of thyroid hormone (T_3) on β receptors in rat brain was observed. Approximately 300 μg of membrane protein was mixed with different concentrations of ~ 3H-DHA. The nonspecific binding tube was additionally treated with racemic propranolol (final concentration 10 -4 M) and the total reaction volume was 25 μl. 30 ℃ incubated for 15 minutes, the reaction was terminated immediately with a domestic glass fiber membrane filtration, with 20ml diluted 1: 3 cold buffer wash, separation and binding of ~ 3H-DHA free. Filter dry xylene scintillation fluid,