Goswami A, Rosenberg I N
Department of Medicine, Boston University School of Medicine, Massachusetts 02118.
Biochem Biophys Res Commun. 1990 Nov 30;173(1):6-12. doi: 10.1016/s0006-291x(05)81013-7.
We have isolated and purified iodothyronine 5'-deiodinase from rat liver microsomes to homogeneity as judged by PAGE and analytical HPLC. The enzyme progressively lost activity after solubilization, and specific activity enhancement was a modest 22-fold, but the final preparation still had substantial activity and was used for molecular characterization. The enzyme had an Mr of 56,000 with a single band in SDS-PAGE, suggesting absence of subunit structure. The high Km, and the GSH-responsive low Km, activities were co-purified, but the low Km enzyme lost GSH-responsiveness upon pretreatment with dithiothreitol (DTT) and urea. The enzyme was strongly inhibited by the iron chelator, alpha,alpha'-dipyridyl and showed a broad absorbance band at 410 nm. Spectral analysis with diethylpyrocarbonate (DEPC) revealed 5 histidine residues/mol enzyme, while enzyme activity was inhibited by DEPC in a pseudo-first order process with modification of 1 histidine residue/mol.
我们已从大鼠肝脏微粒体中分离并纯化出碘甲状腺原氨酸5'-脱碘酶，经聚丙烯酰胺凝胶电泳（PAGE）和分析型高效液相色谱（HPLC）鉴定，该酶已达到均一性。该酶在增溶后活性逐渐丧失，比活性仅适度提高了22倍，但最终制剂仍具有较高活性，并用于分子特性研究。该酶的相对分子质量为56,000，在十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）中呈现单一条带，表明不存在亚基结构。高Km活性和对谷胱甘肽（GSH）敏感的低Km活性可共同纯化，但低Km酶在用二硫苏糖醇（DTT）和尿素预处理后失去了对GSH的敏感性。该酶受到铁螯合剂α,α'-联吡啶的强烈抑制，并在410nm处呈现宽吸收带。用焦碳酸二乙酯（DEPC）进行光谱分析显示，每摩尔酶有5个组氨酸残基，而酶活性在DEPC的作用下以假一级反应过程受到抑制，每摩尔有1个组氨酸残基被修饰。
