O-glycosylation of intact and truncated ribophorins in brefeldin A- treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases

Glycosidase treatment of immunoprecipitated proteins was carried out according to protocols provided by the suppliers. Protein A-sepharose beads carrying the immunoprecipitates were boiled for 2 rain in a buffer containing 0.3% SDS and 100 mM Na-phosphate, pH 6.5. The beads were then sedimented and the supernatanls containing the eluted protein diluted with the same volume of a NP-40 containing buffer to final concentrations of 0.9% NP-40, 10 mM EDTA, 50 mM Na-phosphnte, 50 mM Na-acetate, pH 5.5, 20 U/ml trasylol. Aliquots (50 ~d) were then treated for 24 h (or mock treated) with the following glycosidases before gel electrophoresis: O-glycosidase (endo-N-acetyl-a-D-galactosaminidase from Diplococcus pneumoniae, 2.5 mU/sample), ~5-D-galactosidase (from bovine testes, 5 mU/ sample), endo-O-D-galactusidase (from Bacteriodes frasilis , 5 mU/sampie), C~-L-fucosidase (from beef kidney, 5 mU/sample in a dilation buffer containing 100 mM Na-acetate, pH 4.5, and no Na-phosphate), N-acetyl-O-D-glucosaminidase (from beef kidney, 50 mU/sample) and neuraminidase (from Clostridium perfringens, 5 mU/sample).