Purification and characterization of rat liver nuclear thyroid hormone receptors.
Purification and characterization of rat liver nuclear thyroid hormone receptors.
复制标题
大鼠肝核甲状腺激素受体的纯化和表征。
DOI：
10.1073/pnas.84.10.3420
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发表时间：
1987
影响因子：
11.1
通讯作者：
DeGroot,LJ
中科院分区：
文献类型：
--
作者：
Ichikawa,K;DeGroot,LJ
通过依次使用羟基磷灰石柱层析、硫酸铵沉淀、Sephadex G-150凝胶过滤、DNA-纤维素柱层析、DEAE-Sephadex柱层析和肝素-Sepharose柱层析，将核甲状腺激素受体纯化至每mg蛋白质具有904 pmol的L-3，5，3 ′-三碘甲状腺原氨酸（T3）结合容量，回收率为2.5-5.2%。假设一个T3分子与受体的49，000-Da单位结合，我们从4-5 kg大鼠肝脏中可重复地获得6.4-14.7 μ g受体蛋白，纯度为4.2-4.9%。使用10 mM吡哆醛5 ′-磷酸盐从肝素-琼脂糖凝胶柱上洗脱受体，观察到其减少受体与肝素-琼脂糖凝胶或DNA-纤维素的结合。这种效应对吡哆醛5 '-磷酸盐是特异性的，因为相关化合物无效。纯化的受体以高亲和力（6.0 × 10（9）升/摩尔）结合T3，碘甲腺原氨酸类似物对纯化受体的亲和力顺序与粗品受体制剂中观察到的亲和力顺序相同[3，5，3 '-三碘甲腺原氨酸（D-T3）大于L-T3大于D-3，5，3'-三碘甲腺原氨酸（D-T3）大于L-甲状腺素大于D-甲状腺素]。纯化的受体的沉降系数为3.4 S，斯托克斯半径为34 A，计算分子量为49，000。在NaDodSO 4/聚丙烯酰胺凝胶电泳后通过银染色鉴定的几个条带中，一个49，000-Da蛋白质显示[125 I]甲状腺素的光亲和标记，其可被过量的未标记T3取代。亲和标记受体的胰蛋白酶片段和内源性蛋白酶消化片段分别在27，000-Da和36，000-Da肽中显示饱和结合。这些分子量与凝胶过滤和梯度沉降的估计值一致，表明亲和标记发生在核甲状腺激素受体的激素结合结构域。该方法可重复地以有用的量和纯度提供经典的天然大鼠肝T3核受体，并且具有迄今为止报道的最高比活性。
Nuclear thyroid hormone receptor was purified to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding capacity per mg of protein with 2.5-5.2% recovery by sequentially using hydroxylapatite column chromatography, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column chromatography, DEAE-Sephadex column chromatography, and heparin-Sepharose column chromatography. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, we reproducibly obtained 6.4-14.7 micrograms of receptor protein with 4.2-4.9% purity from 4-5 kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate, which was observed to diminish binding of receptor to heparin-Sepharose or DNA-cellulose. This effect was specific for pyridoxal 5'-phosphate, since related compounds were not effective. Purified receptor bound T3 with high affinity (6.0 X 10(9) liter/mol), and the order of affinity of iodothyronine analogues to purified receptor was identical to that observed with crude receptor preparations [3,5,3'-triiodothyroacetic acid greater than L-T3 greater than D-3,5,3'-triiodothyronine (D-T3) greater than L-thyroxine greater than D-thyroxine]. Purified receptor had a sedimentation coefficient of 3.4 S, Stokes radius of 34 A, and calculated molecular mass of 49,000. Among several bands identified by silver staining after electrophoresis in NaDodSO4/polyacrylamide gels, one 49,000-Da protein showed photoaffinity labeling with [125I]thyroxine that was displaceable with excess unlabeled T3. The tryptic fragment and endogenous proteinase-digested fragment of the affinity-labeled receptor showed saturable binding in 27,000-Da and 36,000-Da peptides, respectively. These molecular masses are in agreement with estimates from gel filtration and gradient sedimentation, indicating that affinity labeling occurred at the hormone binding domain of nuclear thyroid hormone receptor. This procedure reproducibly provides classical native rat liver T3 nuclear receptor in useful quantity and purity and of the highest specific activity so far reported.