Cysteine proteinases from human thyroids and their actions on thyroglobulin.

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.