Synthesis and initial processing of gastric apomucin
Zalesna, G.; Tsukada, H.; Okazaki, K.; Slomiany, B.L.; Slomiany, A.
Biochemistry International 18(4): 775-784
1989
ISSN/ISBN: 0158-5231 PMID: 2764978 Document Number: 345270
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 2,000 Da peptides, whereas N-acetylgalactosamine was discernible on 5,000Da peptides.