Yang Y W, Romanus J A, Liu T Y, Nissley S P, Rechler M M
J Biol Chem. 1985 Feb 25;260(4):2570-7.
BRL-3A rat liver cells synthesize mature 7484-dalton rat insulin-like growth factor II (rIGF-II) as a approximately 22-kDa precursor, presumably prepro-rIGF-II. In the present study, we have biosynthetically labeled intact BRL-3A cells with [35S]cysteine and immunoprecipitated cell lysates and media with antisera to rIGF-II. A approximately 20-kDa protein was identified in immunoprecipitates of cell lysates having properties consistent with pro-rIGF-II. The approximately 20-kDa protein is precipitated by immune sera but not by nonimmune serum. Its immunoprecipitation is specifically inhibited by unlabeled rIGF-II but not by insulin. It is not precipitated from labeled lysates of a subclone of BRL-3A cells (BRL-3A2) that does not synthesize rIGF-II. The approximately 20-kDa protein is rapidly labeled intracellularly (10 min) but is not detected in BRL-3A media. In pulse-chase experiments, radioactivity in the approximately 20-kDa protein disappears during the chase and appears, at later times, in specifically immunoprecipitated approximately 19-, approximately 10-, approximately 8-, and approximately 7-kDa proteins in media and, to a limited extent, intracellularly. A protein with electrophoretic mobility identical to that of the approximately 20-kDa protein observed in cell lysates is immunoprecipitated from 35S-proteins whose synthesis is directed by BRL-3A RNA in a reticulocyte lysate cell-free translation system supplemented with microsomal membranes, and presumably arises by cotranslational removal of the signal peptide from approximately 22-kDa prepro-rIGF-II. Processing of the approximately 20-kDa protein in intact BRL-3A cells to intermediate and mature rIGF-II species appears to occur at the time of secretion and/or shortly thereafter, with the different forms appearing at approximately the same time.
BRL - 3A大鼠肝细胞将成熟的7484道尔顿大鼠胰岛素样生长因子II（rIGF - II）合成为一种约22 kDa的前体，推测为前胰岛素原 - rIGF - II。在本研究中，我们用[35S]半胱氨酸对完整的BRL - 3A细胞进行生物合成标记，并用抗rIGF - II抗血清对细胞裂解物和培养基进行免疫沉淀。在细胞裂解物的免疫沉淀物中鉴定出一种约20 kDa的蛋白质，其性质与胰岛素原 - rIGF - II一致。约20 kDa的蛋白质可被免疫血清沉淀，但不能被非免疫血清沉淀。其免疫沉淀被未标记的rIGF - II特异性抑制，但不被胰岛素抑制。它不能从不合成rIGF - II的BRL - 3A细胞亚克隆（BRL - 3A2）的标记裂解物中沉淀出来。约20 kDa的蛋白质在细胞内迅速被标记（10分钟），但在BRL - 3A培养基中未检测到。在脉冲追踪实验中，约20 kDa蛋白质中的放射性在追踪过程中消失，并在稍后的时间出现在培养基中特异性免疫沉淀的约19 kDa、约10 kDa、约8 kDa和约7 kDa蛋白质中，并且在细胞内也有一定程度的出现。在补充微粒体膜的网织红细胞裂解物无细胞翻译系统中，从由BRL - 3A RNA指导合成的35S蛋白质中免疫沉淀出一种电泳迁移率与在细胞裂解物中观察到的约20 kDa蛋白质相同的蛋白质，推测它是由约22 kDa前胰岛素原 - rIGF - II共翻译去除信号肽产生的。完整的BRL - 3A细胞中约20 kDa蛋白质加工成中间和成熟的rIGF - II形式似乎发生在分泌时和/或之后不久，不同形式大约同时出现。
