den Hartog M T, Sijmons C C, Bakker O, Ris-Stalpers C, de Vijlder J J
Academic Medical Center, University of Amsterdam, The Netherlands.
Eur J Endocrinol. 1995 May;132(5):611-7. doi: 10.1530/eje.0.1320611.
Thyroxine (T4) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T4 formation. In order to investigate the specific role of various tyrosine residues in T4 formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T4 formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T4 formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T4 formation in this mutated N-terminal TG fragment was lower, the ultimate T4 generation was the same as in the wild-type fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
甲状腺素（T4）由甲状腺球蛋白（TG）内碘化酪氨酸残基偶联形成。在成熟的TG中，一些碘化酪氨酸残基优先参与T4的形成。为了研究各种酪氨酸残基在T4形成中的特定作用，构建了具有突变酪氨酸残基的N端TG片段。一个大小为198个氨基酸、在氨基酸位置5、29、89、97、107、130和192处含有七个酪氨酸残基的N端TG片段在杆状病毒系统中表达。使用定点诱变构建了八个突变TG片段，其中不同的酪氨酸残基被苯丙氨酸取代。在前四个TG突变体中，单个酪氨酸残基（5、89、97或130）发生突变。在突变体Y(5,89,97,130)F中，这四个酪氨酸残基全部被取代。第六个突变体Y(29,89,107,130,192)F仅包含酪氨酸残基5和97，第七个（Y(29,89,97,192)F）仅包含酪氨酸残基5、107和130。一个所有酪氨酸残基都被苯丙氨酸取代的TG片段（Y(5,29,89,97,107,130,192)F）用作阴性对照。在用乳过氧化物酶进行体外碘化后，在未突变的野生型N端TG片段中确定了特异性T4的形成。一般来说，当198个氨基酸片段中酪氨酸残基的总数减少时，突变TG构建体中的T4形成减少，但含有三个酪氨酸残基（其中两个是5和130）的片段Y(29,89,97,192)除外。尽管这个突变的N端TG片段中T4形成的速率较低，但最终的T4生成量与野生型片段相同。（摘要截断于250字）
