Spiro M J, Spiro R G
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215.
Endocrinology. 1988 Jul;123(1):56-65. doi: 10.1210/endo-123-1-56.
Thyroglobulin from colloid as well as from membrane fractions became radiolabeled upon incubation of calf thyroid slices with [35S]sulfate. The identity of the sulfate-labeled molecule was established by immunoprecipitation, polyacrylamide gel electrophoresis, Bio-Gel A-5m filtration, and DEAE-cellulose chromatography. Size analysis by gel filtration of [35S]glycopeptides and hydrazine-released oligosaccharides indicated that the sulfate was primarily located in the complex (unit B) carbohydrate units of thyroglobulin. Moreover, although [35S]sulfate-labeled oligosaccharides were cleaved by N-glycanase to the same extent as those labeled with [3H]mannose, they were not released by endo-beta-N-acetylglucosaminidase under conditions that led to the complete removal of polymannose carbohydrate (unit A). The failure of 35S-labeled glycopeptides and oligosaccharides to bind to immobilized Concanavalin-A indicated that the sulfate residues in calf thyroglobulin are located in carbohydrate units with three or more branches. No evidence for the occurrence of tyrosine sulfate was found upon examination of Pronase digests of radiolabeled thyroglobulin, and chemical analyses excluded the presence of this amino acid down to a level of 0.5 residues/polypeptide subunit. Studies with density gradient-separated membrane fractions as well as with puromycin indicated that sulfate addition is a late event in thyroglobulin biosynthesis which occurs in the Golgi compartment. Furthermore, it was observed that the nondimerized thyroglobulin subunit was much less sulfate labeled than the mature molecule. The location of the sulfated carbohydrate in a terminal portion of the calf thyroglobulin peptide chain was suggested by the observation that the subunit [mol wt (Mr) = 330,000] can undergo a transformation, presumably mediated by an endogenous protease, to a sulfate-free component (Mr = approximately 270,000) with the appearance of a 35S-labeled 60,000 Mr fragment; the release of a single sulfate-labeled peptide (Mr = 60,000) by mild trypsin treatment was consistent with a sequestration of sulfate groups in the thyroglobulin molecule.
用[35S]硫酸盐孵育小牛甲状腺切片后，来自胶体以及膜组分的甲状腺球蛋白都被放射性标记。通过免疫沉淀、聚丙烯酰胺凝胶电泳、Bio-Gel A-5m过滤和DEAE-纤维素色谱法确定了硫酸盐标记分子的身份。通过凝胶过滤对[35S]糖肽和肼释放的寡糖进行大小分析表明，硫酸盐主要位于甲状腺球蛋白的复合（B单元）碳水化合物单元中。此外，尽管[35S]硫酸盐标记的寡糖被N-聚糖酶切割的程度与用[3H]甘露糖标记的寡糖相同，但在导致多聚甘露糖碳水化合物（A单元）完全去除的条件下，它们不会被内切β-N-乙酰氨基葡萄糖苷酶释放。35S标记的糖肽和寡糖不能与固定化的伴刀豆球蛋白A结合，这表明小牛甲状腺球蛋白中的硫酸盐残基位于具有三个或更多分支的碳水化合物单元中。在检查放射性标记的甲状腺球蛋白的链霉蛋白酶消化物时，未发现酪氨酸硫酸盐存在的证据，化学分析排除了该氨基酸的存在，其含量低至0.5个残基/多肽亚基。对密度梯度分离的膜组分以及嘌呤霉素的研究表明，硫酸盐添加是甲状腺球蛋白生物合成中的后期事件，发生在高尔基体区室。此外，观察到未二聚化的甲状腺球蛋白亚基的硫酸盐标记比成熟分子少得多。小牛甲状腺球蛋白肽链末端部分存在硫酸化碳水化合物，这一位置是通过观察到亚基[分子量（Mr）=330,000]可以经历一种转变推测而来的，这种转变可能由内源性蛋白酶介导，转变为无硫酸盐成分（Mr =约270,000），同时出现一个35S标记的60,000 Mr片段；温和胰蛋白酶处理释放出单个硫酸盐标记肽（Mr = 60,000），这与甲状腺球蛋白分子中硫酸盐基团的隔离一致。
