Leonard J L, Visser T J, Leonard D M
Department of Cellular and Molecular Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA. jack
J Biol Chem. 2001 Jan 26;276(4):2600-7. doi: 10.1074/jbc.M006973200. Epub 2000 Oct 23.
Type I iodothyronine deiodinase is a approximately 50-kDa, integral membrane protein that catalyzes the outer ring deiodination of thyroxine. Despite the identification and cloning of a 27-kDa selenoprotein with the catalytic properties of the type I enzyme, the composition and the physical nature of the active deiodinase are unknown. In this report, we use a molecular approach to determine holoenzyme composition, the role of the membrane anchor on enzyme assembly, and the contribution of individual 27-kDa subunits to catalysis. Overexpression of an immunologically unique rat 27-kDa protein in LLC-PK1 cells that contain abundant catalytically active 27-kDa selenoprotein decreased deiodination by approximately 50%, and > 95% of the LLC-PK1 derived 27-kDa selenoprotein was specifically immune precipitated by the anti-rat enzyme antibody. The hybrid enzyme had a molecular mass of 54 kDa and an s(20,w) of approximately 3.5 S indicating that every native 27-kDa selenoprotein partnered with an inert rat 27-kDa subunit in a homodimer. Enzyme assembly did not depend on the presence of the N-terminal membrane anchor of the 27-kDa subunit. Direct visualization of the deiodinase dimer showed that the holoenzyme was sorted to the basolateral plasma membrane of the renal epithelial cell.
I型碘甲状腺原氨酸脱碘酶是一种分子量约为50 kDa的整合膜蛋白，可催化甲状腺素的外环脱碘反应。尽管已鉴定并克隆出一种具有I型酶催化特性的27 kDa硒蛋白，但活性脱碘酶的组成和物理性质仍不清楚。在本报告中，我们采用分子方法来确定全酶组成、膜锚定在酶组装中的作用以及单个27 kDa亚基对催化的贡献。在含有大量具有催化活性的27 kDa硒蛋白的LLC-PK1细胞中过表达一种免疫独特的大鼠27 kDa蛋白，可使脱碘反应降低约50%，并且LLC-PK1来源的27 kDa硒蛋白中> 95%可被抗大鼠酶抗体特异性免疫沉淀。杂交酶的分子量为54 kDa，s(20,w)约为3.5 S，这表明每个天然的27 kDa硒蛋白在同型二聚体中与一个无活性的大鼠27 kDa亚基配对。酶组装不依赖于27 kDa亚基N端膜锚定的存在。脱碘酶二聚体的直接可视化显示，全酶被分选到肾上皮细胞的基底外侧质膜。
