Davidson H W, Peshavaria M, Hutton J C
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
Biochem J. 1987 Sep 1;246(2):279-86. doi: 10.1042/bj2460279.
The nature of the endoproteolytic activity involved in the post-translational processing of proinsulin has been investigated in rat insulinoma tissue. 125I-proinsulin was converted by lysed insulin-secretory granules into insulin via an intermediate form identified as des-dibasic-proinsulin. This activity co-localized with immunoreactive (endogenous) insulin and carboxypeptidase H upon subcellular fractionation of the tissue, indicating a secretory-granular location. Under optimized conditions, conversion was quantitative. Inhibitor studies demonstrated that processing occurred by a reaction sequence involving cleavage on the C-terminal side of the pairs of basic amino acids, with subsequent removal of the newly exposed basic residues by carboxypeptidase H. Endoproteolytic activity was abolished by EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not by 1,10-phenanthroline or by group-specific inhibitors of serine, thiol or acidic proteinases. Inhibition by EDTA and CDTA could be reversed by both Ca2+ and Zn2+, although the former appeared to be the ion of physiological importance. Addition of Ca2+ in the absence of chelators stimulated endoproteinase activity, with a maximal effect at 5 mM, a concentration consistent with the intragranular environment. Similarly the pH optimum of 5.5 coincides with the prevailing intragranular pH. Together these properties suggest that the Ca2+-dependent endopeptidase described here is involved in vivo in the proteolytic processing of proinsulin.
已在大鼠胰岛素瘤组织中研究了胰岛素原翻译后加工过程中所涉及的内切蛋白水解活性的性质。125I-胰岛素原通过裂解的胰岛素分泌颗粒转化为胰岛素，中间形式被鉴定为去双碱性胰岛素原。在对该组织进行亚细胞分级分离时，这种活性与免疫反应性（内源性）胰岛素和羧肽酶H共定位，表明其位于分泌颗粒中。在优化条件下，转化是定量的。抑制剂研究表明，加工过程是通过一个反应序列进行的，该序列涉及在一对碱性氨基酸的C末端一侧进行切割，随后通过羧肽酶H去除新暴露的碱性残基。EDTA和CDTA（1,2-环己二胺四乙酸）可消除内切蛋白水解活性，但1,10-菲咯啉或丝氨酸、巯基或酸性蛋白酶的基团特异性抑制剂则不能。EDTA和CDTA的抑制作用可被Ca2+和Zn2+逆转，尽管前者似乎是具有生理重要性的离子。在没有螯合剂的情况下添加Ca2+可刺激内蛋白酶活性，在5 mM时达到最大效果，该浓度与颗粒内环境一致。同样，最适pH值为5.5与颗粒内普遍的pH值相符。这些特性共同表明，本文所述的Ca2+依赖性内肽酶在体内参与胰岛素原的蛋白水解加工过程。
