Transport, function, and sorting of lactase-phlorizin hydrolase in Madin-Darby canine kidney cells.

摘要: Lactase-phlorizin hydrolase (LPH), a small intestinal brush-border glycoprotein, is synthesized as single chain precursor (pro-LPH, M(r) = 215,000-230,000) that undergoes cleavage to the final mature form (LPHm, 160,000 in human). In human and pig intestine well colon carcinoma cell line Caco-2, this takes place intracellularly prior insertion into membrane. To assess role of proteolytic on transport, function, sorting LPH stable Madin-Darby canine kidney was generated which expresses (denoted MDCK-ML). Biosynthetic labeling experiments demonstrated transport kinetics posttranslational processing pattern are similar those cells. Moreover, enzymatic activity found be indistinguishable from (LPHm). The studied by biosynthetic cells grown filters followed surface immunoprecipitation. Here, we could demonstrate cleaved LPHm molecule predominantly at apical membrane, whereas complex glycosylated uncleaved pro-LPH (pro-LPHc) targeted both domains, basolateral. pulse-chase 20 degrees C arrested trans-Golgi network, did not take place. By contrast, when chase temperature raised 37 pro-LPHc resumed, occurred. We conclude post-trans-Golgi network event most likely implicated exposition otherwise masked elements.