SUBCELLULAR LOCALIZATION OF TSH PROCESSING

这项研究的目的是确定亚细胞 甲状腺刺激的翻译后加工的位置 激素（TSH）亚单位前体。 将使用小鼠促甲状腺肿瘤 作为积极分泌促甲状腺激素细胞的来源; 甲状腺功能正常和甲状腺切除的小鼠将是“休息”的来源， “刺激的”促甲状腺激素细胞。 肿瘤和垂体组织切碎 将在体外与[S-35]蛋氨酸、[H-3]岩藻糖、[S-35] 硫酸盐、[H-3]N-乙酰甘露糖胺或[H-3]甘露糖，然后匀浆 并通过我们已经用于 以前的研究。 富含粗面内质网的细胞组分， 近端高尔基体、远端高尔基体和分泌颗粒将被表征 通过电子显微镜，并测定“细胞器特异性”酶，如 作为半乳糖基转移酶。 标记前体的顺序运动 将分析脉冲追踪研究期间这些隔室之间的差异。 将使用羧基氰化物间氯苯腙（CCCP）和莫能菌素 作为亚细胞分离的先驱， 处理事件的亚细胞位置。 标记的TSH亚基将被 从细胞匀浆、细胞组分和培养基中免疫沉淀，和 将通过SDS-聚丙烯酰胺凝胶电泳表征。 [H-3] 甘露糖标记的寡糖将从TSH亚单位释放， 糖苷内切酶H，并通过纸确定其结构 层析 我们将确定这三种组织类型在以下方面是否不同： 新合成的TSH亚单位在亚细胞中的分布 隔室、处理事件的位置或动力学，或 亚基在隔室之间的移位。 我们将为每一个 每种组织类型的区室的α-β的程度和动力学 亚基组合、亚基摩尔比、亚基含量 某些残基（岩藻糖、唾液酸和硫酸盐），以及 高甘露寡糖亚基。 与研究不同， 糖蛋白加工的其他人，我们建议研究一个 免疫反应系统，其中主要的寡糖 分泌的物质，TSH，被认为是重要的生物 活动 我们的研究结果将与罕见的 下丘脑甲状腺功能减退症和垂体促甲状腺激素分泌肿瘤。 主教， 我们希望能对基础细胞生物学有更好的理解 促甲状腺激素的合成。

The objective of the proposed research is to determine the subcellular locations of the post-translational processing of thyroid-stimulating hormone (TSH) subunit precursors. Mouse thyrotropic tumor will be utilized as a source of actively secreting thyrotrophs; pituitary tissue of euthyroid and thyroidectomized mice will be sources of "resting" and "stimulated" thyrotrophs, respectively. Tumor and pituitary tissue minces will be incubated in vitro with [S-35] methionine, [H-3] fucose, [S-35] sulfate, [H-3]N- acetylmannosamine, or [H-3] mannose, and then homogenized and fractionated by the sucrose step-gradient method that we have used for previous studies. Cell fractions enriched in rough endoplasmic reticulum, proximal Golgi, distal Golgi, and secretory granules will be characterized by electron microscopy, and assayed for "organelle-specific" enzymes, such as galactosyltransferase. The sequential movement of labeled precursors between these compartments during pulse-chase studies will be analyzed. Carboxyl cyanide m-chlorophenylhydrazone (CCCP) and monensin will be used as adjuncts to subcellular fractionation to make inferences about the subcellular location of processing events. Labeled TSH subunits will be immunoprecipitated from cell homogenates, cell fractions, and media, and will be characterized by SDS-polyacrylamide gel electrophoresis. [H-3] mannose-labeled oligosaccharides will be released from TSH subunits by endoglycosidase H, and their structures determined by paper chromatography. We will determine whether the three tissue types differ in their distribution of newly synthesized TSH subunits among subcellular compartments, the locations or kinetics of processing events, or the translocation of subunits between compartments. We will determine for each compartment of each tissue type the extent and kinetics of alpha-beta subunit combination, the molar ratios of subunits, the subunit content of certain residues (fucose, sialic acid, and sulfate), and the structures of the high mannose oligosaccharides of subunits. Unlike studies of glycoprotein processing by others, we propose to study a hormonally-responsive system in which the oligosaccharides of a major secreted species, TSH, are believed to be important for biological activity. Our findings will be clinically relevant to rare patients with hypothalamic hypothyroidism and pituitary TSH-producing tumors. Primarily, we hope to contribute to a better understanding of the basic cell biology of TSH biosynthesis.