Biosynthesis and intracellular transport of rat liver 5'-nucleotidase.
To investigate biosynthesis and intracellular transport of 5'-nucleotidase, we purified this enzyme from rat liver and prepared antibodies. In immunoblot analysis, 5'-nucleotidase from the plasmalemmal, Golgi, and tritosomal fractions migrated as a single band of 72 kDa. The immunoreactive 68-kDa band was detected in the rough microsomal fraction and only the 72-kDa polypeptide contained sialic acids. The single polypeptide of 61 kDa immunoprecipitated from the translation products with the membrane-bound polysomal RNAs by the cell-free system converted to the 68-kDa form associated with membranes, when translated in the presence of microsomal vesicles. 5'-Nucleotidase from cultured primary hepatocytes pulse-labeled with [35S]methionine for 20 min migrated as a 68-kDa polypeptide. Within 45 min of chase, the 68-kDa form was converted to the 72-kDa form. Upon treatment with tunicamycin, a new immunoreactive polypeptide of 59 kDa was obtained. These results suggest that 5'-nucleotidase is translated on the membrane-bound polysomes as a 61-kDa precursor and that the cleavage of the 2-kDa signal peptide and the core glycosylation co-translationally convert the precursor to the 68-kDa form, which is subsequently processed in the Golgi complex to the 72-kDa form.
D015720:5'-Nucleotidase; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport; D002478:Cells, Cultured; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D019158:N-Acetylneuraminic Acid; D009708:Nucleotidases / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021382:Protein Sorting Signals / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012794:Sialic Acids / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D021382:Protein Sorting Signals; D012794:Sialic Acids; D014415:Tunicamycin; D009708:Nucleotidases; D015720:5'-Nucleotidase; D019158:N-Acetylneuraminic Acid