Abstract
The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-3H]palmitic acid and [3H]proline. The mucus glycoprotein polymer, secreted into the growth medium (extracellular) and that contained within the cells (intracellular), was purified from the other components of the secretion, thoroughly delipidated, and then analyzed for the radiolabeled tracers. Both pools of mucus glycoprotein, incubated in the presence of [3H]palmitic acid, contained radioactive label which could not be removed by gel filtration, CsCl density gradient centrifugation, sodium dodecyl sulfate-gel electrophoresis, or lipid extraction. Treatment of the purified mucus glycoprotein with 1 M hydroxylamine or 0.3 M methanolic KOH released the radioactivity, thus indicating that [3H]palmitic acid was covalently bound by ester linkage to the glycoprotein. The released radioactivity was associated mainly (87%) with palmitic acid. The incorporation ratio of [3H]proline to [3H]palmitic acid was 0.12:1.0 in the extracellular glycoprotein and 1.38:1.0 in the intracellular glycoprotein, which suggested that acylation of mucus glycoprotein occurs in the intracellular compartment after completion of its polypeptide core. The fact that incorporation of [3H]palmitic acid was greater in the glycoprotein subunits than in the glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer.
Highlights
The incorporation of fatty acids into gastric mucus glycoproteins was studied by incubating rat gastric mucosal cell suspensions with [9,10-'H]palmitic acid and ['Hlproline
The fact that incorporation of ['Hlpalmitic acid was greater in the glycoprotein subunits than in the'glycoprotein polymer indicates that acylation takes place near the end of subunit processing but before their assembly into the high molecular weight mucus glycoprotein polymer
To determine if and when fatty acid attachment occurs during glycoprotein processing and secretion, we have subjected the rat gastric mucosal cell suspensions to a short-term cell culture and thenanalyzed the mucus glycoprotein isolated from extracellularand intracellular pools for the content of radiolabeled ester-bound fatty acids
Summary
The results presented inthis study provide evidence for the covalent attachment of fatty acids to gastric mucus glycoprotein synthesized in uitro. SDS2-polyacrylamidegel electrophoresis, column chromatography on Bio-Gel A-50, and CsCl density gradient centrifugation of the secretory products of cultured mucosal cells showed that [3H]palmitic acid was incorporated only into the mucus glycoprotein polymer and its precursor subunits. The incorporation of [3H] proline, an amino acid characteristic of mucus glycoproteins, into the secretory products of the mucosal cells was less selective and the label was found in several other fractions. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
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