Transport and metabolism of 5'-nucleotidase in a rat hepatoma cell line.
The biosynthesis of the ectoenzyme 5'-nucleotidase in the rat hepatoma cell line H4S has been studied by pulse-labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5'-Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS-gels of 72 kDa. The enzyme is initially synthesized as a 68-kDa precursor which is converted to the mature 72-kDa form in 15-60 min (t1/2 = 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N-glycosylation, revealed six species of 5'-nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N-linked oligosaccharide chains accounting for the difference between the 58-kDa polypeptide backbone and the 68-kDa species. The 68-kDa precursor is susceptible to cleavage by endo-beta-N-acetylglycosaminidase H; the 72-kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5'-nucleotidase keeps one or two high-mannose or hybrid chains in the mature form, even after prolonged pulse-chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20-30 min. The half-life of 5'-nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5'-nucleosidase is released into the culture medium.
D015720:5'-Nucleotidase; D000118:Acetylglucosaminidase; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D007120:Immunochemistry; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009708:Nucleotidases / Q000378:metabolism*; D051381:Rats
D009708:Nucleotidases; D015720:5'-Nucleotidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase