Dunn A D, Dunn J T
Department of Medicine, University of Virginia School of Medicine, Charlottesville 22908.
Endocrinology. 1988 Aug;123(2):1089-97. doi: 10.1210/endo-123-2-1089.
This report describes properties of highly purified cathepsin-B and an additional cysteine proteinase, designated cysteine proteinase I, obtained from human thyroids. Both enzymes are localized to lysosomes. The activity profile of cysteine proteinase I combined with its sensitivity to the active site inhibitor Z-Phe-Phe-CNH2 suggest that it is distinct from other cysteine proteinases described so far. Cysteine proteinase I and cathepsin-B had respective pH optima of 3.5-4.0 and 4.5-5.0 with thyroglobulin (Tg) as substrate. Based on Km/Kcat (catalytic constant) ratios, cysteine proteinase I degraded rabbit [125I]Tg to peptide intermediates 50 times more efficiently than did cathepsin-B. Under conditions of limited digestion, both enzymes cleaved Tg at three or more sites, producing iodinated fragments of 20,000-50,000 mol wt (cysteine proteinase I) or 10,000-40,000 mol wt (cathepsin-B). Tryptic digests of these fragments were isolated by HPLC, and those containing thyroid hormone were sequenced for identification of amino acids and localization of 125I. Cysteine proteinase I cleaved peptides primarily from the C-terminal region of Tg, which contained two major hormonogenic sites, while cathepsin-B produced peptides mainly from the N-terminus, containing another major hormonogenic site. We suggest that the roles of cysteine proteinase I and cathepsin-B are the rapid initial fragmentation of Tg at opposite ends of the molecule, making hormone-containing sites accessible to additional cleavage by other lysosomal endopeptidases and exopeptidases.
本报告描述了从人甲状腺中获得的高度纯化的组织蛋白酶B和另一种半胱氨酸蛋白酶（称为半胱氨酸蛋白酶I）的特性。这两种酶都定位于溶酶体。半胱氨酸蛋白酶I的活性谱及其对活性位点抑制剂Z-Phe-Phe-CNH2的敏感性表明，它与迄今为止描述的其他半胱氨酸蛋白酶不同。以甲状腺球蛋白（Tg）为底物时，半胱氨酸蛋白酶I和组织蛋白酶B的最适pH分别为3.5 - 4.0和4.5 - 5.0。基于Km/Kcat（催化常数）比值，半胱氨酸蛋白酶I将兔[125I]Tg降解为肽中间体的效率比组织蛋白酶B高50倍。在有限消化条件下，两种酶都在三个或更多位点切割Tg，产生分子量为20,000 - 50,000（半胱氨酸蛋白酶I）或10,000 - 40,000（组织蛋白酶B）的碘化片段。通过高效液相色谱法分离这些片段的胰蛋白酶消化产物，并对含有甲状腺激素的片段进行测序以鉴定氨基酸并定位125I。半胱氨酸蛋白酶I主要从Tg的C末端区域切割肽，该区域包含两个主要的激素生成位点，而组织蛋白酶B主要从N末端产生肽，其中包含另一个主要的激素生成位点。我们认为，半胱氨酸蛋白酶I和组织蛋白酶B的作用是在分子的相对两端对Tg进行快速的初始片段化，使含激素的位点能够被其他溶酶体内切肽酶和外切肽酶进一步切割。
