Biosynthesis of alpha-galactosidase A in cultured Chang liver cells.
An investigation of the structure and biosynthesis of alpha-galactosidase A (alpha-D-galactoside glycohydrolase, EC 3.2.1.22) and its N-linked oligosaccharide chains was undertaken by metabolic labeling of Chang liver cells with [2-3H]mannose, immunoprecipitation of the activity, and examination of the resulting immunoprecipitates. From cells pulse labeled for 3 h, two radioactive bands with Mr = 58,000 and 49,000 were detected by SDS-gel electrophoresis; following a 20-h chase, only the Mr = 49,000 band was observed. Examination of the oligosaccharide fraction derived from pulse-labeled enzyme revealed that 18% of the asparagine-linked oligosaccharides were complex and 82% were high-mannose type. After a 20-h chase, 48% of the oligosaccharides were complex and 52% were high mannose. The high-mannose oligosaccharides of alpha-galactosidase A immunoprecipitated from both pulsed and pulse-chased cells had the same mobilities as Man8-9GlcNAc on thin-layer chromatography and Bio-Gel P-4. Two fractions of complex glycopeptides derived from the alpha-galactosidase A of pulsed and pulse-chased cells had the same migration on Bio-Gel P-4 as glucose oligomers containing 14 and 19-39 glucose units. Based on their apparent size and their behavior on concanavalin A-Sepharose, the complex oligosaccharides are believed to be composed of tri- and/or tetraantennary structures.
D000818:Animals; D002478:Cells, Cultured; D011232:Chemical Precipitation; D002845:Chromatography; D005696:Galactosidases / Q000096:biosynthesis*; D006801:Humans; D007106:Immune Sera; D007120:Immunochemistry; D008099:Liver / Q000201:enzymology*; D009844:Oligosaccharides / Q000302:isolation & purification; D011817:Rabbits; D000519:alpha-Galactosidase / Q000096:biosynthesis*
D007106:Immune Sera; D009844:Oligosaccharides; D005696:Galactosidases; D000519:alpha-Galactosidase