Schoenmakers C H, Pigmans I G, Visser T J
Department of Internal Medicine III, Erasmus University Medical School, Rotterdam, The Netherlands.
Mol Cell Endocrinol. 1995 Feb;107(2):173-80. doi: 10.1016/0303-7207(94)03440-5.
In the present study the hypothesis was tested that N-bromoacetyl-3,3',5-[125I]triiodothyronine (BrAc[125I]T3) is a useful affinity label for both type I and type III iodothyronine deiodinases (ID-I and ID-III). Therefore, the microsomal fractions of various rat tissues were tested for ID-I and ID-III activities, and microsomal proteins were labeled with BrAc[125I]T3 and analyzed by SDS-PAGE. In agreement with previous observations, high ID-I activities were found in liver, kidney and thyroid, and high ID-III activities in brain, in particular fetal brain, and placenta. SDS-PAGE of BrAc[125I]T3-labeled microsomes showed a prominent radioactive approximately 27 kDa protein (p27) in liver, kidney and thyroid, which was previously identified as ID-I, and a approximately 32 kDa protein (p32) in brain, in particular fetal brain, and placenta. A good correlation was found between the affinity labeling of p32 and the inactivation of ID-III by BrAcT3, suggesting that p32 represents ID-III or a subunit thereof. After treatment of microsomes with 0.05% deoxycholate or carbonate buffer (pH 11.5) p32 was still labeled by BrAc[125I]T3, indicating that p32 is a transmembrane protein. Although 3,3',5'-triiodothyronine (rT3) is not a substrate for ID-III, p32 was readily labeled with BrAc[125I]rT3. Labeling of p32 in rat brain microsomes by BrAc[125I]rT3 was not affected by addition of 100 microM unlabeled thyroxine (T4) or T3, whereas deiodination of [125I]T3 by ID-III was inhibited by 91 and 96% in the presence of 1 microM T4 and T3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
在本研究中，对N-溴乙酰基-3,3',5-[125I]三碘甲状腺原氨酸（BrAc[125I]T3）是否是I型和III型碘甲状腺原氨酸脱碘酶（ID-I和ID-III）的有效亲和标记物这一假设进行了验证。因此，对各种大鼠组织的微粒体部分进行了ID-I和ID-III活性检测，并用BrAc[125I]T3标记微粒体蛋白，然后通过SDS-PAGE进行分析。与先前的观察结果一致，在肝脏、肾脏和甲状腺中发现了高ID-I活性，在脑，尤其是胎儿脑和胎盘中发现了高ID-III活性。用BrAc[125I]T3标记的微粒体进行SDS-PAGE分析显示，在肝脏、肾脏和甲状腺中有一种约27 kDa的突出放射性蛋白（p27），其先前被鉴定为ID-I，在脑，尤其是胎儿脑和胎盘中有一种约32 kDa的蛋白（p32）。发现p32的亲和标记与BrAcT3对ID-III的失活之间存在良好的相关性，这表明p32代表ID-III或其一个亚基。用0.05%脱氧胆酸盐或碳酸盐缓冲液（pH 11.5）处理微粒体后，p32仍能被BrAc[125I]T3标记，这表明p32是一种跨膜蛋白。虽然3,3',5'-三碘甲状腺原氨酸（rT3）不是ID-III的底物，但p32很容易被BrAc[125I]rT3标记。BrAc[125I]rT3对大鼠脑微粒体中p32的标记不受添加100 microM未标记甲状腺素（T4）或T3的影响，而在1 microM T4和T3存在的情况下，ID-III对[125I]T3的脱碘作用分别被抑制了91%和96%。（摘要截短于250字）
