Comparisons of N-linked carbohydrate on platelet (plt) and AtT 20 cell-derived multimerin. Cells were labeled for 28 hours as in Fig 6B; their multimerin radioimmunoprecipitates were treated with buffer, N-glycosidase F (endo-F), or endoglycosidase H (endo-H) and then separated using reduced 4% to 8% SDS-PAGE (lane L, multimerin constitutively secreted during the labeling; lane C, multimerin remaining in the cell lysate postchase; lane S, longer exposure showing the multimerin secreted postchase in response to 8-Br-cAMP). The125I-labeled platelet multimerin subunits p-155 (arrows) and p-170 (arrowheads) are indicated. The coprecipitated, 83-kD platelet protein, which was resistant to N-glycosidase F and N-glycosidase H, was not observed in all studies. The multimerin stored in AtT 20 cells was more extensively proteolyzed than constitutively secreted multimerin. Two of the multimerin subunits constitutively secreted by AtT 20 cells (lanes L) comigrated with p-155 and p-170 after their N-linked carbohydrates were removed with N-glycanase F. The major form of multimerin stored in AtT 20 cells (lanes C) was a proteolytic product smaller than platelet multimerin, although AtT 20 cells also stored a subunit that comigrated with p-155 after N-glycosidase F treatment. Endoglycosidase H-sensitive carbohydrate was detected on the mature multimerin stored and secreted from AtT 20 cells, but not on platelet multimerin.
