Stannard B S, Gesundheit N, Ronin C, Burnside J, Weintraub B D
Molecular, Cellular, and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
J Biol Chem. 1988 Jun 15;263(17):8309-17.
In pulse-chase experiments we compared the kinetics of early carbohydrate processing and subsequent secretion of thyroid-stimulating hormone (TSH) and free alpha subunit under control conditions and after treatment with 1-deoxynojirimycin, an inhibitor of glucosidases I and II. Under control conditions TSH achieved resistance to endo-beta-N-acetylglucosaminidase H (endo H) more rapidly than free alpha (t1/2 0.3 h versus 0.9 h); however, free alpha was secreted more rapidly than TSH (t1/2 2.2 h versus 3.4 h). With 1-deoxynojirimycin, oligosaccharides co-migrating with G3Man9GlcNAc and G2Man9GlcNAc were demonstrated on TSH for the first time, suggesting that previous pulse-chase studies did not disclose these intermediates due to rapid removal of glucose residues from the common G3Man9GlcNAc2 precursor. 1-Deoxynojirimycin delayed the rate of attainment of endo H resistance for both TSH and free alpha, but there was no effect on subunit combination. With 5 mM 1-deoxynojirimycin the amount of secreted free alpha was reduced to 65% of control; secreted TSH was reduced markedly to 17% of control without intracellular accumulation, suggesting increased intracellular degradation. There was no significant toxicity from these doses of 1-deoxynojirimycin on the production or secretion of the two major nonglycosylated pituitary proteins, growth hormone and prolactin, or on at least 10 other secretory proteins. Basal differences in the relative rates of TSH and free alpha processing and secretion as well as differential sensitivity to 1-deoxynojirimycin suggest separate secretory pathways for these two closely related proteins.
在脉冲追踪实验中，我们比较了在对照条件下以及用1-脱氧野尻霉素（一种葡糖苷酶I和II的抑制剂）处理后，甲状腺刺激激素（TSH）和游离α亚基早期碳水化合物加工及随后分泌的动力学。在对照条件下，TSH比游离α亚基更快获得对内切β-N-乙酰葡糖胺酶H（endo H）的抗性（半衰期分别为0.3小时和0.9小时）；然而，游离α亚基的分泌比TSH更快（半衰期分别为2.2小时和3.4小时）。使用1-脱氧野尻霉素时，首次在TSH上发现了与G3Man9GlcNAc和G2Man9GlcNAc共迁移的寡糖，这表明先前的脉冲追踪研究由于从常见的G3Man9GlcNAc2前体中快速去除葡萄糖残基而未揭示这些中间体。1-脱氧野尻霉素延迟了TSH和游离α亚基获得endo H抗性的速率，但对亚基组合没有影响。使用5 mM 1-脱氧野尻霉素时，分泌的游离α亚基量降至对照的65%；分泌的TSH显著降至对照的17%，且无细胞内积累，这表明细胞内降解增加。这些剂量的1-脱氧野尻霉素对两种主要的非糖基化垂体蛋白、生长激素和催乳素的产生或分泌，以及至少10种其他分泌蛋白没有明显毒性。TSH和游离α亚基加工及分泌相对速率的基础差异以及对1-脱氧野尻霉素的不同敏感性表明这两种密切相关蛋白有独立的分泌途径。
