Safran M, Köhrle J, Braverman L E, Leonard J L
Department of Medicine, University of Massachusetts Medical Center, Worcester 01655.
Endocrinology. 1990 Feb;126(2):826-31. doi: 10.1210/endo-126-2-826.
Type I iodothyronine 5'deiodinase (5'D-I) is a membrane-bound enzyme catalyzing the deiodination of T4 to T3. The affinity label, N-bromoacetyl-thyroxine (BrAcT4), has previously been used to characterize a 27 kilodalton protein (p27) from rat liver and kidney microsomes with characteristics of the catalytic subunit of the 5'D-I. We examined the effect of physiological conditions, known to alter 5'D-I activity, on affinity-labeled proteins in rat liver and kidney microsomes. To confirm that the affinity labeled protein was associated with the deiodinase, we treated rats with the active site directed enzyme inhibitor, propylthiouracil (PTU), in the absence and presence of 100-fold excess methimazole (MMI), an antithyroid drug which blocks PTU inhibition of 5'D-I in vivo. In addition, we used the affinity label as a probe to measure 5'D-I levels in membrane preparations from short and long term fasted rats. Rats were treated ip with PTU (50 micrograms/100 g BW) or MMI (5 mg/100 g BW); in a second experiment, groups of rats were fasted for 4 days (4 D), 1 day (1 D), or fed ad lib (C) and hepatic and kidney microsomes were prepared. 5'D-I activity and 5'D-I content, as judged by specific incorporation of the affinity label into p27, were determined. PTU decreased both 5'D-I activity and BrACT incorporation into p27 by 60-65%. Coadministration of MMI attenuated the effect of PTU on 5'D-I activity and p27 affinity labeling. No other affinity labeled proteins were affected. In fasting experiments, the changes in affinity labeling of p27 paralleled the changes in 5'D-I activity. 5'D-I activity was significantly decreased in hepatic microsomes obtained from 4 D-fasted rats as compared to C rats, but was unchanged in hepatic microsomes from 1 D-fasted rats or in kidney microsomes from 1 D or 4 D-fasted rats as compared to C. Maximal BrAcT4 incorporation into p27 decreased by 45% in hepatic microsomes from 4 D-starved rats as compared to C (6.7 +/- 0.9 vs. 11.9 +/- 1.5 pmol BrAcT4 incorporated/mg microsomal protein, respectively). There was no change in p27 content in hepatic microsomes from 1 D-starved rats (11.2 +/- 1.1). Starvation failed to alter the BrAcT4 labeling of kidney microsomes (16.7 +/- 4.4, 16.2 +/- 6.6, and 14.8 +/- 3.2 pmol BrAcT4 in 4 D, 1 D, and C rats, respectively). In this study, we have demonstrated that alterations in biological activity of 5'D-I correspond to alterations in affinity labeling of p27.
I型碘甲状腺原氨酸5'-脱碘酶（5'D-I）是一种膜结合酶，催化T4脱碘生成T3。亲和标记物N-溴乙酰甲状腺素（BrAcT4）先前已用于鉴定大鼠肝脏和肾脏微粒体中一种27千道尔顿的蛋白质（p27），其具有5'D-I催化亚基的特征。我们研究了已知会改变5'D-I活性的生理条件对大鼠肝脏和肾脏微粒体中亲和标记蛋白的影响。为了证实亲和标记的蛋白与脱碘酶相关，我们在不存在和存在100倍过量甲巯咪唑（MMI）（一种抗甲状腺药物，可在体内阻断PTU对5'D-I的抑制作用）的情况下，用活性位点导向的酶抑制剂丙硫氧嘧啶（PTU）处理大鼠。此外，我们使用亲和标记物作为探针来测量短期和长期禁食大鼠膜制剂中的5'D-I水平。大鼠腹腔注射PTU（50微克/100克体重）或MMI（5毫克/100克体重）；在第二个实验中，将大鼠分组禁食4天（4D）、1天（1D）或自由进食（C），并制备肝脏和肾脏微粒体。通过亲和标记物特异性掺入p27来判断5'D-I活性和5'D-I含量。PTU使5'D-I活性和BrACT掺入p27的量均降低了60 - 65%。联合给予MMI减弱了PTU对5'D-I活性和p27亲和标记的影响。没有其他亲和标记的蛋白受到影响。在禁食实验中，p27亲和标记的变化与5'D-I活性的变化平行。与C组大鼠相比，从禁食4天的大鼠获得的肝脏微粒体中5'D-I活性显著降低，但与C组相比，禁食1天的大鼠肝脏微粒体或禁食1天或4天的大鼠肾脏微粒体中的5'D-I活性没有变化。与C组相比，禁食4天的大鼠肝脏微粒体中最大BrAcT4掺入p27的量降低了45%（分别为6.7±0.9与11.9±1.5 pmol BrAcT4掺入/毫克微粒体蛋白）。禁食1天的大鼠肝脏微粒体中p27含量没有变化（11.2±1.1）。饥饿未能改变肾脏微粒体的BrAcT4标记（分别为4D、1D和C组大鼠中的16.7±4.4、16.2±6.6和14.8±3.2 pmol BrAcT4）。在本研究中，我们证明了5'D-I生物学活性的改变与p27亲和标记的改变相对应。
