One step isolation of bovine asialoglycoprotein receptor and its characterization by sequence analysis and MALDI mass spectrometry

The asialoglycoprotein receptor (ASGP-R), which is responsible for the upta ke of partially deglycosylated serum glycoproteins was isolated from bovine liver. The receptor was purified in one step from solubilized plasma membr anes by affinity chromatography on 6-(beta-D-lactosyl)-n-hexylamine coupled to N-hydroxysuccinimide activated Sepharose with a coupling degree of 7.6 mu mol/ml gel. The preparation yielded two distinct polypeptides with appar ent molecular weights of 48 and 43 kDa as determined by sodium dodecyl sulf ate-polyacrylamide gel electrophoresis. A polyclonal antibody raised agains t the human ASGP-R recognized the bovine 43 kDa protein in Western blot ana lysis. The 48 and 43 kDa polypeptides were digested by trypsin and the dige sts were subsequently analyzed by matrix-assisted laser desorption/ionizati on time-of-flight (MALDI-TOF) mass spectrometry. Sequence analysis of four tryptic fragments, two each of the 48 kDa and of the 43 kDa polypeptides re vealed that these were highly homologous to ASGP-R subunits from man, mouse and rat.