Taurog A, Dorris M L
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Arch Biochem Biophys. 1991 Jun;287(2):288-96. doi: 10.1016/0003-9861(91)90481-w.
A recent paper (Buchberger, W., 1988, J. Chromatogr. 432, 57) on lactoperoxidase-catalyzed bromination of tyrosine and thyroglobulin stated, without evidence, that thyroid peroxidase (TPO) is able to use bromide as a substrate. This was in disagreement with unpublished experiments previously performed in this laboratory, and we undertook, therefore, to examine this subject further. Highly purified porcine TPO was compared with lactoperoxidase (LPO) and chloroperoxidase (CPO) for ability to catalyze bromination of tyrosine, thyroglobulin, and bovine serum albumin (BSA). The incubation mixture contained 50-100 nM peroxidase, 10-500 microM 82Br-, tyrosine (150 microM), thyroglobulin (0.3 or 1 microM), or BSA (7.5 microM), and a source of H2O2. The latter was either generated by glucose (1 mg/ml)-glucose oxidase (0.5 or 1 micrograms/ml), or added initially as a bolus (100 microM). With TPO, formation of organically bound 82Br was undetectable under all conditions in the pH range 5.4-7.0. Lactoperoxidase and CPO, on the other hand, displayed considerable brominating activity. Lactoperoxidase was much more active at pH 5.4 than at pH 7.0 and was more active with BSA as acceptor than with tyrosine or thyroglobulin. The distribution of 82Br among the various amino acids in LPO-brominated thyroglobulin and BSA was determined by HPLC. As expected, monobromotyrosine and dibromotyrosine together comprised the greatest part of the bound 82Br. However, a surprisingly high percentage (20-25%) was present as monobromohistidine. Evidence was also obtained for the presence of a small percentage of the bound 82Br as tetrabromothyronine. Peroxidase-catalyzed bromination probably depends on the oxidation of Br- to Br+ by the Compound I form of the enzyme. Since oxidation of Br- to Br+ requires a stronger oxidant than oxidation of I- to I+, our results suggest that Compound I of LPO and of CPO has a higher oxidation potential than Compound I of TPO. In vivo experiments with rats on a low iodine diet injected with 82Br- showed that even under conditions of high stimulation by thyrotropic hormone, there is negligible formation of organic bromine in the thyroid. Measurements of thyroid:serum concentration ratios for 82Br- in similar rats provided no evidence that Br- is a substrate for the iodide transport system of the thyroid.
最近一篇关于乳过氧化物酶催化酪氨酸和甲状腺球蛋白溴化反应的论文（布赫贝格尔，W.，1988年，《色谱杂志》432卷，57页）未经证实就指出，甲状腺过氧化物酶（TPO）能够将溴化物用作底物。这与本实验室之前未发表的实验结果不一致，因此，我们着手进一步研究这个问题。将高度纯化的猪TPO与乳过氧化物酶（LPO）和氯过氧化物酶（CPO）进行比较，以考察它们催化酪氨酸、甲状腺球蛋白和牛血清白蛋白（BSA）溴化反应的能力。孵育混合物含有50 - 100 nM过氧化物酶、10 - 500 μM 82Br-、酪氨酸（150 μM）、甲状腺球蛋白（0.3或1 μM）或BSA（7.5 μM），以及过氧化氢源。后者要么由葡萄糖（1 mg/ml）-葡萄糖氧化酶（0.5或1 μg/ml）产生，要么最初作为一次注射量添加（100 μM）。对于TPO，在pH值5.4 - 7.0的所有条件下均未检测到有机结合82Br的形成。另一方面，乳过氧化物酶和CPO表现出相当可观的确溴化活性。乳过氧化物酶在pH 5.4时比在pH 7.0时活性高得多，并且以BSA作为受体时比以酪氨酸或甲状腺球蛋白时活性更高。通过高效液相色谱法测定了LPO溴化的甲状腺球蛋白和BSA中各种氨基酸之间82Br的分布。正如预期的那样，一溴酪氨酸和二溴酪氨酸合起来占结合82Br的最大部分。然而，令人惊讶的是，有相当高的百分比（20 - 25%）以一溴组氨酸的形式存在。也获得了证据表明有一小部分结合82Br以四溴甲状腺原氨酸的形式存在。过氧化物酶催化的溴化反应可能取决于酶的化合物I形式将Br-氧化为Br+。由于将Br-氧化为Br+比将I-氧化为I+需要更强的氧化剂，我们的结果表明LPO和CPO的化合物I比TPO的化合物I具有更高的氧化电位。对低碘饮食的大鼠注射82Br-进行的体内实验表明，即使在促甲状腺激素高度刺激的条件下，甲状腺中有机溴的形成也可忽略不计。对类似大鼠中82Br-的甲状腺：血清浓度比的测量没有提供证据表明Br-是甲状腺碘转运系统的底物。
