Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography.
Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography.
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DOI：
10.1083/jcb.80.3.715
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发表时间：
1979-03
期刊：
影响因子：
--
通讯作者：
Orci L
中科院分区：
文献类型：
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作者：
Habener JF;Amherdt M;Ravazzola M;Orci L
甲状旁腺中甲状旁腺激素（PTH）的形成是通过两个连续的蛋白水解裂解发生的，裂解来自较大的生物合成前体。PTH mRNA编码的初始产物是甲状旁腺激素（pre- proparyroid hormone, preproth），一种115个氨基酸的多肽。在合成的1分钟内，由于从pre - proth中去除nh2末端的25个氨基酸，形成了多肽，甲状旁腺激素（proth）。延迟15-20分钟后，去除proth的nh2端6个氨基酸序列，得到84个氨基酸的PTH。为了研究甲状旁腺细胞中生物合成前体进行特异性蛋白水解裂解的亚细胞位点，我们通过电子显微镜放射自显影检查了放射自显影颗粒的时空迁移，并通过电泳检查了用[3H]亮氨酸孵育5分钟（脉冲孵育），然后用未标记的亮氨酸孵育85分钟（追逐孵育），观察牛甲状旁腺切片中标记的pre - proth消失和标记的proth向PTH转化的动力学。5分钟时，85%的放射自显影颗粒局限于粗内质网（RER）。培养15 min后，高尔基区放射自显影颗粒数量迅速增加；15 ~ 30分钟内，它们在仍位于高尔基体区的分泌囊内迁移，然后迁移到高尔基体区外的成熟分泌颗粒。电泳分析表明，pre - proth迅速消失（5分钟），在15分钟时首次检测到proth向PTH的转化，并在30分钟内完成。在孵育后期（30-90分钟），分泌颗粒内的放射自显影颗粒迁移到细胞周围和质膜，与30分钟首次检测到的甲状旁腺激素释放相关。我们得出结论，pre - proth到proth的蛋白水解转化发生在内质网中，随后的proth到PTH的转化发生在高尔基复合体中。
The formation of parathyroid hormone (PTH) in the parathyroid gland occurs via two successive proteolytic cleavages from larger biosynthetic precursors. The initial product coded for by PTH mRNA is pre-proparathyroid hormone (PreProPTH), a polypeptide of 115 amino acids. Within 1 min of synthesis, the polypeptide, proparathyroid hormone (ProPTH), is formed as a result of the proteolytic removal of the NH2-terminal 25 amino acids from Pre-ProPTH. After a delay of 15-20 min, the NH2-terminal six-amino acid sequence of ProPTH is removed to give PTH of 84 amino acids. To investigate the subcellular sites in the parathyroid cell where the biosynthetic precursors undergo specific proteolytic cleavages, we examined, by electron microscopy autoradiography, the spatiotemporal migration of autoradiographic grains and, by electrophoresis, the kinetics of the disappearance of labeled Pre-ProPTH and the conversion of labeled ProPTH to PTH in bovine parathyroid gland slices incubated with [3H]leucine for 5 min (pulse incubation) followed by incubations with unlabeled leucine for periods up to 85 min (chase incubations). By 5 min, 85% of the autoradiographic grains were confined to the rough endoplasmic reticulum (RER). Autoradiographic grains increased rapidly in number in the Golgi region after 15 min of incubation; from 15 to 30 min they migrated within secretory vesicles still in the Golgi region and then migrated to mature secretory granules outside the Golgi area. Electrophoretic analyses showed that Pre-ProPTH disappeared rapidly (by 5 min) and that conversion of ProPTH to PTH was first detectable at 15 min and was completed by 30 min. At later times of incubation (30-90 min), autoradiographic grains within the secretion glanules migrated to the periphery of the cell and to the plasma membrane, in correlation with the release of PTH first detected by 30 min. We conclude that proteolytic conversion of Pre-ProPTH to ProPTH takes place in the RER and that subsequent conversion of ProPTH to PTH occurs in the Golgi complex.