MacGregor R R, Jilka R L, Hamilton J W
J Biol Chem. 1986 Feb 5;261(4):1929-34.
Monolayer cultures of bovine parathyroid cells or fresh gland slices were incubated with radioactive amino acids in order to study the formation and metabolism of parathormone (PTH). PTH, secretory protein I, and COOH-terminal fragments of PTH were all released into media within 30 min, most strongly in the first hour after synthesis. Peptides in tissue, cells, and media were separated using reverse-phase high performance liquid chromatography. In eluates of media, six radioactive peaks were prominent. The first four and the sixth were immunoreactive in a COOH-terminal specific PTH radioimmunoassay, but only the sixth was reactive in an NH2-terminal specific assay. Under conditions where recovery of PTH(1-34) was quantitative, gel filtration of media was used to show that no NH2-terminal fragments of PTH were secreted. Sequence analyses of secreted COOH-terminal peptides indicated that the NH2 termini of the first three peaks corresponded to residues 43, 37, and 34 of PTH. The fourth peak contained a mixture of two peptides with NH2 termini at residues 24 and 28 of PTH. The fifth could not be identified; the sixth was PTH. Cleavages at the 23-24 bond of PTH occurred within minutes of the formation of PTH itself, and the other peptides were formed more slowly. Mandatory cleavage of PTH at the 23-24 peptide bond would destroy the biological activity of the hormone on kidney and bone, a situation consistent with the possibility that intracellular PTH metabolism participates in secretory regulation. The results showed that different peptides were generated in parathyroid cells than were previously shown to be produced by cathepsin B or D. The results suggest that the proteolytic pathway which results in the secretion of PTH fragments is nonlysosomal in nature.
为了研究甲状旁腺激素（PTH）的形成和代谢，将牛甲状旁腺细胞的单层培养物或新鲜腺体切片与放射性氨基酸一起孵育。PTH、分泌蛋白I和PTH的羧基末端片段在30分钟内全部释放到培养基中，在合成后的第一小时释放最为强烈。使用反相高效液相色谱法分离组织、细胞和培养基中的肽。在培养基洗脱液中，六个放射性峰很突出。前四个峰和第六个峰在羧基末端特异性PTH放射免疫测定中具有免疫反应性，但只有第六个峰在氨基末端特异性测定中具有反应性。在PTH(1-34)回收率为定量的条件下，对培养基进行凝胶过滤以显示没有分泌PTH的氨基末端片段。对分泌的羧基末端肽进行序列分析表明，前三个峰的氨基末端对应于PTH的第43、37和34位残基。第四个峰包含两种肽的混合物，其氨基末端位于PTH的第24和28位残基处。第五个峰无法鉴定；第六个峰是PTH。PTH在23-24键处的切割在PTH自身形成后几分钟内发生，其他肽形成得更慢。PTH在23-24肽键处的强制切割会破坏该激素对肾脏和骨骼的生物活性，这种情况与细胞内PTH代谢参与分泌调节的可能性一致。结果表明，甲状旁腺细胞中产生的肽与先前显示由组织蛋白酶B或D产生的肽不同。结果表明，导致PTH片段分泌的蛋白水解途径本质上是非溶酶体的。
