Studies on the attachment of fatty acid and N-acetylgalactosamine to mucus glycoprotein nascent chains in rat gastric mucosa

The authors have investigated the process of mucin protein acylation and glycosylation to ascertain whether fatty acids and galactosamine are added to nascent chains prior to the completion or after release from the ribosome. For fatty acid and carbohydrate labeling, the rat gastric mucosal cell suspension was incubated with [3H] palmitic acid, and [14C]-N-acetylgalactosamine for 3h at 370C. After incubation, the cells were washed with incubation medium and subjected to subcellular fractionation. The peptidyl-tRNA was released from polysomes by sedimentation in sucrose gradient containing EDTA (20-30% sucrose gradient). The fraction containing peptidyl-tRNA was dialyzed and nascent chains were separated by ion-exchange chromatography on DEAE-Sephadex. The peptidyl-tRNA, eluted with a linear NaCl gradient was hydrolyzed and separated by SDS-gel electrophoresis and thin-layer chromatography. The individual bands isolated from thin layer plates were analyzed for the content of [3H] palmitic acid and [14C] N-acetylgalactosamine. The results revealed that fatty acids are incorporated into nascent chains prior to chain completion. Gel electrophoresis and thin layer chromatography showed that the fatty acid moiety is added soon after the acylation site is synthesized on the ribosome, while the GalNAc becomes incorporated only to the acylated peptide