Synthesis and initial processing of gastric apomucin.

The initiation of the processing of apomucin was investigated using mucus glycoprotein synthesizing polysomes from rat gastric epithelial cells. The polysomes were isolated from cells labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine, purified on Helix pomatia-Sepharose affinity column, dissociated to release peptidyl-tRNA, and chromatographed on DEAE-HPLC column to separate peptidyl-tRNA complexes from the free and ribosomal RNA and proteins. The analysis of the HPLC purified peptidyl-tRNA revealed that complexes were labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine. Digestion of the peptidyl-tRNA with RNase released 3H and 14C labeled peptides, while alkaline degradation destroyed the complex and rendered the [3H]palmitic acid extractable with hexane. The treatment of the 3H and 14C labeled peptidyl-tRNA complexes with alpha-N-acetylgalactosaminidase led to the release of radiolabeled N-acetylgalactosamine, whereas alkaline borohydride reduction produced N-acetylgalactosaminitol. The fatty acid residues have been detected in peptidyl-tRNA containing 2,000Da peptides, whereas N-acetylgalactosamine was discernible on 5,000Da peptides.