The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes

Abstract

The synthesis and degradation of 5′-nucleotidase has been studied in rat hepatocytes. Primary cultures of rat hepatocytes were established with the cells showing evidence of polarity after 24–36 h in culture. After a 30 h lag period 5′-nucleotidase activity increased to a plateau level similar to the activity found in whole liver. The half life of the enzyme after reaching the plateau of activity was 22.8 h. Pulse-chase biosynthetic labelling studies of 5′-nucleotidase in the cultured hepatocytes using [ 35S]methionine showed that the 5′-nucleotidase monomer was synthesised as an M r 67 000 form which was converted to the mature M r 72 000 form. [ 35S]Methionine labelling studies in the presence of tunicamycin showed that the unglycosylated protein monomer was an M r 57 000 form. The immature M r 67 000 form of 5′-nucleotidase was sensitive to endoglycosidase H, whereas the mature form was sensitive only to endoglycosidase F. The data presented are consistent with 5′-nucleotidase in a polarised cell being synthesised and processed like other membrane glycoproteins, in contrast to earlier reports.