Casciola-Rosen L A, Hubbard A L
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1992 Apr 25;267(12):8213-21.
We used lactoperoxidase-mediated iodination to investigate the lumenal polypeptide composition of rat hepatocyte endosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase that binds specifically to hepatocyte asialoglycoprotein receptors was perfused through isolated rat livers at 16 degrees C in the presence of mannan, resulting in the accumulation of ligand in early endosomes. Endosome containing low density vesicle fractions were subsequently isolated from sucrose gradients of microsomes, and the lactoperoxidase moiety was used to catalyze the iodination of lumenal-facing proteins. After gel electrophoresis, 125I-labeled early endosomes reproducibly showed a distinct 125I-polypeptide profile containing prominently labeled bands migrating at 43, 52, 58, 90, 110, 135, 230, and greater than 300 kDa. The asialoglycoprotein receptor (43-, 52-, and 58-kDa subunits) was by far the predominantly labeled protein even when iodinations were performed under conditions of receptor-ligand dissociation, and we conclude that it is the most abundant hepatocyte early endosomal protein. Furthermore, the iodination profile of the three asialoglycoprotein receptor subunits differed strikingly from their chemical amounts. Using immunoprecipitation, we directly identified the Na+,K(+)-ATPase; to our knowledge, this is the first biochemical evidence for the Na+,K(+)-ATPase in rat hepatocyte early endosomes. We also directly identified receptors for mannose 6-phosphate, epidermal growth factor, transferrin, and polymeric IgA in 125I-labeled early endosomes.
我们采用乳过氧化物酶介导的碘化法来研究大鼠肝细胞内体的腔内多肽组成。将去唾液酸血清类黏蛋白与乳过氧化物酶的化学偶联物（其可特异性结合肝细胞去唾液酸糖蛋白受体）在16℃下、存在甘露聚糖的情况下灌注通过分离的大鼠肝脏，导致配体在早期内体中积累。随后从微粒体的蔗糖梯度中分离出含有低密度囊泡组分的内体，并且利用乳过氧化物酶部分催化面向腔内的蛋白质的碘化反应。凝胶电泳后，125I标记的早期内体可重复性地显示出独特的125I多肽图谱，其中包含在43、52、58、90、110、135、230和大于300 kDa处迁移的显著标记条带。即使在受体 - 配体解离的条件下进行碘化反应，去唾液酸糖蛋白受体（43 -、52 - 和58 - kDa亚基）仍是标记最主要的蛋白质，并且我们得出结论，它是最丰富的肝细胞早期内体蛋白。此外，三种去唾液酸糖蛋白受体亚基的碘化图谱与其化学计量显著不同。利用免疫沉淀法，我们直接鉴定出了Na +,K(+) - ATP酶；据我们所知，这是大鼠肝细胞早期内体中存在Na +,K(+) - ATP酶的首个生化证据。我们还在125I标记的早期内体中直接鉴定出了甘露糖6 - 磷酸、表皮生长因子、转铁蛋白和聚合IgA的受体。
