Guest P C, Arden S D, Bennett D L, Clark A, Rutherford N G, Hutton J C
University of Cambridge, Department of Clinical Biochemistry, Addenbrookes Hospital, United Kingdom.
J Biol Chem. 1992 Nov 5;267(31):22401-6.
Proinsulin conversion in the insulin secretory granule is mediated by two sequence-specific endoproteases related to the Kex2 homologues, PC2 and PC3 (Bennett, D. L., Bailyes, E. M., Nielsen, E., Guest, P. C., Rutherford, N. G., Arden, S. D., and Hutton, J. C. (1992) J. Biol. Chem. 267, 15229-15236; Bailyes, E. M., Bennett, D. L., and Hutton, J. C. (1992) Enzyme, in press). Radiolabeling studies using isolated rat islets showed that PC2 was synthesized initially as a 76-kDa glycoprotein which was converted by limited proteolysis to the mature 64-66-kDa form. Conversion was initiated approximately 1 h after synthesis and proceeded via intermediates of 71, 68, and 66 kDa with a t1/2 of 140 min. Release of only the 66- and 64-66-kDa radiolabeled forms of PC2 was induced by glucose and then only at times more than 2 h following synthesis. Proinsulin conversion, by contrast, was more rapid (delay = 30 min, t1/2 = 60 min), and release commenced as soon as 1 h after synthesis with the secreted material being comprised of the precursor, intermediate, and mature forms of insulin. Ultrastructural analysis of islet beta cells showed that PC2 was concentrated in secretory granules. Subcellular fractionation combined with immunoblot analysis showed that insulinoma secretory granules contained only the mature 64-66-kDa form of PC2, whereas fractions enriched in Golgi and endoplasmic reticulum contained a mixture of the 76- and 66-kDa forms of the enzyme. These results indicate that post-translational proteolysis of PC2 is initiated before sorting into the regulated pathway of secretion and that the relative proportions of proinsulin and PC2 packaged into secretory granules will change with physiological conditions.
胰岛素分泌颗粒中的胰岛素原转化由两种与Kex2同源物相关的序列特异性内切蛋白酶介导，即PC2和PC3（贝内特，D. L.，贝利耶斯，E. M.，尼尔森，E.，格斯特，P. C.，卢瑟福，N. G.，阿登，S. D.，以及赫顿，J. C.（1992年）《生物化学杂志》267卷，15229 - 15236页；贝利耶斯，E. M.，贝内特，D. L.，以及赫顿，J. C.（1992年）《酶》，即将发表）。使用分离的大鼠胰岛进行的放射性标记研究表明，PC2最初作为一种76 kDa的糖蛋白合成，通过有限的蛋白水解作用转化为成熟的64 - 66 kDa形式。转化在合成后约1小时开始，通过71、68和66 kDa的中间体进行，半衰期为140分钟。只有66 kDa和64 - 66 kDa放射性标记形式的PC2在葡萄糖诱导下释放，且仅在合成后超过2小时释放。相比之下，胰岛素原转化更快（延迟 = 30分钟，半衰期 = 60分钟），合成后1小时就开始释放，分泌的物质包括胰岛素的前体、中间体和成熟形式。胰岛β细胞的超微结构分析表明，PC2集中在分泌颗粒中。亚细胞分级分离结合免疫印迹分析表明，胰岛素瘤分泌颗粒仅含有成熟的64 - 66 kDa形式的PC2，而富含高尔基体和内质网的分级分离物含有该酶的76 kDa和66 kDa形式的混合物。这些结果表明，PC2的翻译后蛋白水解在分选进入调节性分泌途径之前就已开始，并且包装到分泌颗粒中的胰岛素原和PC2的相对比例会随生理条件而变化。
