Visser T J, Kaplan M M, Leonard J L, Larsen P R
J Clin Invest. 1983 Apr;71(4):992-1002. doi: 10.1172/jci110854.
We have studied 5'-deiodination of thyroxine (T(4)) and 3,3',5'-triiodothyronine (rT(3)) in rat pituitary tissue in vitro, with respect to substrate specificity, reaction kinetics, effects of 6-n-propyl-2-thiouracil (PTU), and the time course of effects of thyroid hormone depletion and repletion. Removal of one phenolic iodine or both tyrosyl iodines from the T(4) molecule resulted in compounds that were not deiodinated, but alterations in the alanine side chain had little effect.5'-Deiodination of 2 nM rT(3) by pituitary microsomes from euthyroid rats was inhibited >90% by 1 mM PTU, but was inhibited <10% by 100 nM T(4). The apparent Michaelis constant (K(m)) and maximum velocity (V(max)) for rT(3) at 20 mM dithiothreitol (DTT) were 33 nM and 84 pmol/mg protein per h. This reaction followed ping-pong type reaction kinetics when concentrations of DTT were varied. PTU inhibition was competitive with DTT and uncompetitive with rT(3). In contrast, when pituitary microsomes from hypothyroid rats (21 d postthyroidectomy) were used, deiodination of 2 nM rT(3) was inhibited only 20% by 1 mM PTU and up to 80% by 100 nM T(4). At 20 mM DTT, the apparent K(m) and V(max) in hypothyroid microsomes were 4.7 nM rT(3) and 16 pmol/mg protein per h. T(4) was a competitive inhibitor of PTU-insensitive rT(3) 5'-deiodination (K(i) = 1.3 nM). T(4) 5'-deiodination by hypothyroid microsomes was not affected by PTU, was competitively inhibited by rT(3) (K(i), 1.7 nM), and exhibited sequential type reaction kinetics with DTT as cosubstrate. When T(4) 5'-deiodination was measured in euthyroid and hypothyroid microsomes, respectively, the apparent K(m) and V(max) for T(4) at 20 mM DTT, were 0.9 nM and 0.55 pmol/mg protein per h (euthyroid), and 0.8 nM and 6.9 pmol/mg protein per h (hypothyroid). The T(4) 5'-deiodination rate and the PTU-insensitive, but not total, rT(3) 5'-deiodination rate (i.e. measured in the presence and the absence of 1 mM PTU, respectively) in pituitary homogenates were significantly elevated 24 h after thyroidectomy. PTU-insensitive activity continued to increase until at >/=30 d after thyroidectomy it was 11 times the PTU-insensitive activity in controls. At the latter time, PTU-sensitive rT(3) 5'-deiodinase activity appeared to be decreased. The increase in PTU-insensitive T(4) and rT(3) 5'-deiodination observed 48 h after thyroidectomy was prevented by replacement doses of T(4) or T(3). The PTU-insensitive activity of long term hypothyroid pituitaries was decreased by 71% and >/=84% 4 h after injection of 20 and 200 mug T(3), respectively, with no change in PTU-sensitive rT(3) deiodination. These data show that rat pituitary tissue contains two distinct iodothyronine 5'-deiodinating pathways that differ with respect to substrate specificity, PTU sensitivity, reaction kinetics, and regulation by thyroid hormone. One of these resembles the 5'-deiodinase of liver and kidney, and predominates in euthyroid pituitary tissue in vitro. The other, also found in rat brain, predominates in hypothyroid pituitary tissue, is rapidly responsive to changes in thyroid hormone availability, and, as judged by previous, in vivo studies, appears to account for all the T(3) produced locally in the pituitary and, thereby, 50% of the intracellular T(3) in this tissue.
我们在体外研究了大鼠垂体组织中甲状腺素（T₄）和3,3',5'-三碘甲腺原氨酸（rT₃）的5'-脱碘作用，涉及底物特异性、反应动力学、6-正丙基-2-硫氧嘧啶（PTU）的影响以及甲状腺激素耗竭和补充的时间效应。从T₄分子上去除一个酚羟基碘或两个酪氨酸碘会产生不能脱碘的化合物，但丙氨酸侧链的改变影响很小。正常甲状腺大鼠垂体微粒体对2 nM rT₃的5'-脱碘作用被1 mM PTU抑制>90%，但被100 nM T₄抑制<10%。在20 mM二硫苏糖醇（DTT）存在下，rT₃的表观米氏常数（Kₘ）和最大反应速度（Vₘₐₓ）分别为33 nM和84 pmol/mg蛋白每小时。当改变DTT浓度时，该反应遵循乒乓型反应动力学。PTU抑制作用与DTT呈竞争性，与rT₃呈非竞争性。相反，当使用甲状腺功能减退大鼠（甲状腺切除术后21天）的垂体微粒体时，2 nM rT₃的脱碘作用仅被1 mM PTU抑制20%，被100 nM T₄抑制高达80%。在20 mM DTT时，甲状腺功能减退微粒体中的表观Kₘ和Vₘₐₓ分别为4.7 nM rT₃和16 pmol/mg蛋白每小时。T₄是PTU不敏感的rT₃ 5'-脱碘作用的竞争性抑制剂（抑制常数Kᵢ = 1.3 nM）。甲状腺功能减退微粒体对T₄的5'-脱碘作用不受PTU影响，被rT₃竞争性抑制（Kᵢ，1.7 nM），并且以DTT作为共底物表现出有序型反应动力学。分别在正常甲状腺和甲状腺功能减退微粒体中测定T₄的5'-脱碘作用时，在20 mM DTT存在下，T₄的表观Kₘ和Vₘₐₓ分别为0.9 nM和0.55 pmol/mg蛋白每小时（正常甲状腺），以及0.8 nM和6.9 pmol/mg蛋白每小时（甲状腺功能减退）。甲状腺切除术后24小时，垂体匀浆中T₄的5'-脱碘速率以及PTU不敏感但非总rT₃的5'-脱碘速率（即分别在有和没有1 mM PTU存在下测定）显著升高。PTU不敏感活性持续增加直至甲状腺切除术后≥30天，此时其是对照组PTU不敏感活性的11倍。在后者时间，PTU敏感型rT₃ 5'-脱碘酶活性似乎降低。甲状腺切除术后48小时观察到的PTU不敏感的T₄和rT₃ 5'-脱碘增加可通过补充剂量的T₄或T₃来预防。注射20和200 μg T₃后4小时，长期甲状腺功能减退垂体的PTU不敏感活性分别降低71%和≥84%，而PTU敏感型rT₃脱碘作用无变化。这些数据表明，大鼠垂体组织含有两种不同的碘甲腺原氨酸5'-脱碘途径，它们在底物特异性、PTU敏感性、反应动力学以及甲状腺激素调节方面存在差异。其中一种类似于肝脏和肾脏的5'-脱碘酶，在体外正常甲状腺垂体组织中占主导。另一种也存在于大鼠脑中，在甲状腺功能减退垂体组织中占主导，对甲状腺激素可用性的变化快速响应，并且根据先前的体内研究判断，似乎占垂体局部产生的所有T₃的比例，从而占该组织细胞内T₃的50%。
