Albert S G, Permutt M A
J Biol Chem. 1979 May 10;254(9):3483-92.
The purpose of these experiments was to determine whether insulin-related peptides, larger than proinsulin, could be detected in pancreatic islet cells. Catfish pancreatic islets were incubated with radiolabeled amino acids. After 15- to 60-min incubation, two acid-alcohol-extractable peptides, larger than proinsulin, were detected which were approximately of Mr = 12,000 and 11,000 (12 K and 11K, respectively). They migrated as single polypeptide chains by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis under reducing conditions, and were therefore not aggregates of insulin or proinsulin. The 12 K protein had identical mobility with catfish preoproinsulin synthesized in a wheat germ cell-free system. On standard electrophoresis at pH 8.9, the 12 K protein migrated separately from proinsulin and was at least 65% one protein with two to three minor contaminants. The 12 K and 11 K proteins were chemically related to insulin and proinsulin as shown by tryptic peptide analysis, using cation exchange resin chromatography, and by two-dimensional tryptic peptide maps. Analysis of the tryptic digest of the 12 K protein, compared to proinsulin after leucine aminopeptidase treatment, suggested that the NH2 terminus of the larger protein was different from that of proinsulin. These peptides were specifically bound to anti-insulin antibody. The binding was only 5 to 8% of the protein added, but was specific for the 12 K and 11 K proteins when the immunoprecipitates were examined by electrophoresis and not from contaminating proinsulin. During the continuous incubation of the islets with [3H]leucine, 12 K and 11 K proteins were synthesized in the cell before proinsulin. When islets were first incubated with [3H]leucine for 30 min followed by incubation with excess unlabeled leucine, the 12 K and 11 K proteins appeared to show a precursor-product relationship to proinsulin and insulin. Even when total islet protein synthesis was inhibited by cycloheximide (100 microgram/ml), proinsulin continued to be synthesized for up to 2 h. This suggested that the conversion of the proinsulin precursors to proinsulin in the fish is a post-translational event.
这些实验的目的是确定在胰岛细胞中是否能检测到比胰岛素原更大的胰岛素相关肽。将鲶鱼胰岛与放射性标记的氨基酸一起孵育。孵育15至60分钟后，检测到两种比胰岛素原更大的酸 - 醇可提取肽，其分子量约为12,000和11,000（分别为12K和11K）。在还原条件下，通过十二烷基硫酸钠 - 尿素聚丙烯酰胺凝胶电泳，它们以单条多肽链形式迁移，因此不是胰岛素或胰岛素原的聚集体。12K蛋白与在无细胞小麦胚系统中合成的鲶鱼前胰岛素原具有相同的迁移率。在pH 8.9的标准电泳中，12K蛋白与胰岛素原分开迁移，并且至少65%是一种蛋白质，含有两到三种微量污染物。通过胰蛋白酶肽分析、阳离子交换树脂色谱法以及二维胰蛋白酶肽图谱表明，12K和11K蛋白在化学上与胰岛素和胰岛素原相关。与亮氨酸氨肽酶处理后的胰岛素原相比，对12K蛋白的胰蛋白酶消化分析表明，较大蛋白质的NH2末端与胰岛素原不同。这些肽能特异性结合抗胰岛素抗体。结合量仅为添加蛋白的5%至8%，但当通过电泳检查免疫沉淀物时，这种结合对12K和11K蛋白具有特异性，而不是来自污染的胰岛素原。在用[3H]亮氨酸连续孵育胰岛的过程中，12K和11K蛋白在细胞中先于胰岛素原合成。当胰岛先用[3H]亮氨酸孵育30分钟，然后再用过量未标记的亮氨酸孵育时，12K和11K蛋白似乎与胰岛素原和胰岛素呈现前体 - 产物关系。即使当总胰岛蛋白合成被放线菌酮（100微克/毫升）抑制时，胰岛素原仍能持续合成长达2小时。这表明在鱼类中胰岛素原前体向胰岛素原的转化是一个翻译后事件。
