Khan M N, Savoie S, Bergeron J J, Posner B I
J Biol Chem. 1986 Jun 25;261(18):8462-72.
A protocol employing discontinuous sucrose gradient centrifugation was developed to prepare light mitochondrial (L) and Golgi fraction endosomes from simultaneously prepared parent L and microsomal fractions. As judged by the concentration of labeled hormone postinjection, L intermediate and heavy endosome subfractions were 40- to 175-fold purified and Golgi intermediate and heavy endosome subfractions were 30- to 45-fold purified. On electron microscopy, L endosomal fractions contained a predominance of lipoprotein-filled vesicles and were less heterogeneous than corresponding Golgi endosomal fractions. All endosomal fractions were enriched in receptors for insulin and prolactin but binding sites for the former were more broadly distributed in other subfractions than those for the latter. On Percoll gradient centrifugation, L endosomal fractions yielded one peak (rho 1.057) corresponding to the heavier of two peaks seen in Golgi endosomal fractions. The protein composition of high density L and Golgi endosomes, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was similar. The bulk of marker enzymes assayed did not migrate with the endosomal components. Combined acid phosphatase cytochemistry and electron microscope radioautography established that about 80% of the L endosomes contained no acid phosphatase. By affinity labeling and immunological titration with insulin receptor antibody, insulin receptors were identical in L and Golgi endosomes. Insulin-stimulable receptor kinase was demonstrable in both L and Golgi endosome fractions. Following in vivo insulin administration, the insulin receptor kinase in both L and Golgi endosomes was significantly activated. This activated state was not inhibited by a large excess of antiserum to insulin and thus not due to insulin contaminating the partially purified receptor preparation. These observations are compatible with the maintenance and/or initiation of hormone-dependent phosphorylations intracellularly.
开发了一种采用不连续蔗糖梯度离心的方案，用于从同时制备的亲本轻线粒体（L）和微粒体组分中制备轻线粒体（L）和高尔基体部分内体。根据注射后标记激素的浓度判断，L中间和重内体亚组分纯化了40至175倍，高尔基体中间和重内体亚组分纯化了30至45倍。在电子显微镜下，L内体组分主要包含充满脂蛋白的囊泡，并且比相应的高尔基体内体组分的异质性更低。所有内体组分都富含胰岛素和催乳素受体，但前者的结合位点在其他亚组分中的分布比后者更广泛。在Percoll梯度离心中，L内体组分产生一个峰（密度1.057），对应于高尔基体内体组分中看到的两个峰中较重的峰。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳评估，高密度L和高尔基体内体的蛋白质组成相似。所检测的大部分标记酶没有与内体组分一起迁移。联合酸性磷酸酶细胞化学和电子显微镜放射自显影表明，约80%的L内体不含酸性磷酸酶。通过亲和标记和用胰岛素受体抗体进行免疫滴定，L和高尔基体内体中的胰岛素受体是相同的。L和高尔基体内体组分中均可检测到胰岛素刺激的受体激酶。体内给予胰岛素后，L和高尔基体内体中的胰岛素受体激酶均被显著激活。这种激活状态不受大量过量的胰岛素抗血清的抑制，因此不是由于污染部分纯化受体制剂的胰岛素所致。这些观察结果与细胞内激素依赖性磷酸化的维持和/或启动相一致。
