Leonard J L, Ekenbarger D M, Frank S J, Farwell A P, Koehrle J
Molecular Endocrinology Laboratory, University of Massachusetts Medical School, Worcester 01655.
J Biol Chem. 1991 Jun 15;266(17):11262-9.
Type I iodothyronine 5'-deiodinase is an integral membrane protein catalyzing the phenolic ring deiodination of thyroxine. We recently showed that the substrate binding subunit of this approximately 50-kDa protein is selectively labeled with N-bromoacetyl-L-thyroxine, allowing ready identification of the type I enzyme without the need to maintain catalytic activity. In this study, we used both affinity labeling and catalytic activity to determine the regional distribution of this enzyme in rat kidney and to localize the enzyme to specific plasma membrane domain(s) of renal epithelial cells. The type I enzyme was present exclusively in tubular epithelial cells of the outer renal cortex and co-purified with basolateral plasma membranes; the renal medulla lacked activity. LLC-PK1 cells, derived from the proximal convoluted tubule, have abundant type I 5'-deiodinating activity. We used this homogenous cell line to verify that the type I enzyme was localized to the cytosolic surface of the basolateral membrane. Digitonin permeabilization increased affinity labeling of the enzyme 4-fold, and approximately 75% of the affinity label was incorporated into the 27-kDa substrate binding subunit. Affinity labeling of the type I enzyme in LLC-PK1 cells mimicked the affinity labeling of the substrate binding subunit of type I 5'-deiodinase in rat kidney (Köhrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155-6163). Subcellular fractionation of LLC-PK1 cell homogenates showed that both affinity labeled and catalytically active type I enzyme were present on the cytosolic surface of the basolateral region of the renal cell membrane.
I型碘甲状腺原氨酸5'-脱碘酶是一种催化甲状腺素酚环脱碘的整合膜蛋白。我们最近发现，这种约50 kDa蛋白的底物结合亚基可被N-溴乙酰-L-甲状腺素选择性标记，无需保持催化活性即可轻松鉴定I型酶。在本研究中，我们利用亲和标记和催化活性来确定该酶在大鼠肾脏中的区域分布，并将该酶定位到肾上皮细胞的特定质膜结构域。I型酶仅存在于肾外皮质的肾小管上皮细胞中，并与基底外侧质膜共纯化；肾髓质缺乏活性。源自近端曲管的LLC-PK1细胞具有丰富的I型5'-脱碘活性。我们使用这种同质细胞系来验证I型酶定位于基底外侧膜的胞质表面。洋地黄皂苷通透化使该酶的亲和标记增加了4倍，约75%的亲和标记掺入到27 kDa的底物结合亚基中。LLC-PK1细胞中I型酶的亲和标记模拟了大鼠肾脏中I型5'-脱碘酶底物结合亚基的亲和标记（Köhrle, J., Rasmussen, U. B., Ekenbarger, D. M., Alex, S., Rokos, H., Hesch, R. D., and Leonard, J. L. (1990) J. Biol. Chem. 265, 6155 - 6163）。LLC-PK1细胞匀浆的亚细胞分级分离表明，亲和标记的和具有催化活性的I型酶均存在于肾细胞膜基底外侧区域的胞质表面。
