Köhrle J, Rasmussen U B, Rokos H, Leonard J L, Hesch R D
Abteilung Klinische Endokrinologie, Medizinische Hochschule, Hannover, Federal Republic of Germany.
J Biol Chem. 1990 Apr 15;265(11):6146-54.
125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.
用125I标记的L-甲状腺素和L-三碘甲状腺原氨酸的N-溴乙酰衍生物作为烷基化亲和标记物，以鉴定大鼠肝脏和肾脏微粒体膜上特异性结合甲状腺激素的蛋白质。通过乙醇沉淀分析亲和标记物的掺入情况，并在还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离后，通过放射自显影鉴定各个亲和标记的蛋白质。溴乙酰-L-甲状腺素（BrAcT4）和溴乙酰-L-三碘甲状腺原氨酸（BrAcT3）均能亲和标记6至8种大小在17至84 kDa之间的膜蛋白。亲和标记具有时间和温度依赖性，还原型二硫醇和去污剂均可增加亲和标记，主要作用于一种27 kDa的蛋白质。在最佳条件下，高达80%的亲和标记与一种27 kDa的蛋白质（p27）相关联。0.4 nM BrAc[125I]L-T4对p27的亲和标记可被0.1 μM的烷基化配体BrAcT4、BrAcT3或100 μM碘乙酸盐阻断，也可被10 μM浓度的非烷基化可逆配体N-乙酰-L-甲状腺素、3,3',5'-三碘甲状腺原氨酸、3,5-二碘水杨酸酯以及T4拮抗黄酮类化合物EMD 21388阻断。10 μM的L-T4、10 μM的N-乙酰三碘甲状腺原氨酸或10 μM的L-三碘甲状腺原氨酸均不能阻断p27或其他亲和标记条带的亲和标记。过量的烷基化配体BrAcT4、BrAcT3和碘乙酸盐可部分抑制17 kDa条带的亲和标记，但其他小条带的标记不会被过量的竞争者阻断。尽管观察到相似的条带模式，但BrAc[125I]T4产生的亲和标记掺入量高于BrAc[125I]T3，只是BrAcT3对肝脏中58,000 Da的条带和肾脏中53,000 - 55,000 Da的条带亲和标记更强。以p27为主条带的其他亲和标记蛋白质的模式在肝脏和肾脏中相似。通过化学裂解和蛋白酶切割对亲和标记的p27和p55条带进行肽图谱分析，未发现共同条带，排除了p27是p55降解产物的可能性。这些数据表明，T4和T3的N-溴乙酰衍生物亲和标记了有限但相似的一组膜蛋白，且BrAcT4的掺入量大于BrAcT3。在与测量甲状腺激素5'-脱碘相同的条件下，一种低丰度（2 - 5 pmol/mg微粒体蛋白）且具有反应性巯基基团的膜蛋白（p27）被选择性标记。只有p27在存在非共价结合的抑制剂或底物时，对5'-脱碘酶表现出差异亲和标记，这表明p27可能是大鼠肝脏和肾脏中I型5'-脱碘酶的一个组成部分。
