Biosynthesis and secretion of alpha 1-antitrypsin in primary cultures of rat hepatocytes. Characterization of differently glycosylated intracellular and extracellular forms.
The biosynthesis and secretion of alpha 1-antitrypsin was studied in rat hepatocyte primary cultures. After labeling with [35S]methionine an alpha 1-antitrypsin with an apparent molecular weight of 49000 estimated by sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis was immunoprecipitated from the cell homogenate. This intracellular form of alpha 1-antitrypsin could be deglycosylated by endoglycosidase H treatment indicating that its oligosaccharide chains were of the high-mannose type. Pulse-chase experiments showed that about 30 min after its synthesis the transformation of the 49000-Mr alpha 1-antitrypsin to a protein with an apparent molecular weight of 54000 began. Only this 54000-Mr protein was secreted by the hepatocytes. The 54000-Mr alpha 1-antitrypsin was not sensitive to endoglycosidase H, but sensitive to neuraminidase, and it incorporated [3H]galactose and [3H]fucose indicating that its oligosaccharide chains were of the complex type. In the presence of tunicamycin, which blocks the formation of N-asparagine-linked oligosaccharide chains, an unglycosylated alpha 1-antitrypsin with an apparent molecular weight of 41000 was found in the cells as well as in the medium. However, tunicamycin decreased the secretion of alpha 1-antitrypsin by 60-70%, whereas the secretion of albumin remained unaffected. In the presence of colchicine the secretion of both alpha 1-antitrypsin and albumin was impaired. The results demonstrate the importance of glycosylation for the secretion of alpha 1-antitrypsin.
D000818:Animals; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D005260:Female; D008099:Liver / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014415:Tunicamycin / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000378:metabolism*
D000515:alpha 1-Antitrypsin; D014415:Tunicamycin; D003078:Colchicine