1. d-[2−14C]Glucose, [2−14C]acetate, hydroxy[3−14C]pyruvate, [3−14C]pyruvate and [U−14C]glycine were incorporated by surviving scrapings of sheep colonic mucosal tissue into glycoprotein. 2. d-[2−14C]Glucose, [2−14C]acetate, incorporated hydroxy-[3−14C]pyruvate and [3−14C]pyruvate resulted in labelling of each of the monosaccharide residues of the glycoprotein, namely N-glycollylneuraminic acid, N-acetylneuraminic acid, galactose, fucose, glucosamine and galactosamine. [U−14C]Glycine was incorporated as glycyl and seryl residues of the glycoprotein. 3. Despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollylneuraminic acid and N-acetylneuraminic acid were 8·5 and 5·2% by weight of the glycoprotein respectively) the corresponding ratio of the radio-active labelling from d-[2−14C]glucose in N-glycollylneuraminic acid to that in N-acetylneuraminic acid was 1·00:7·27 (expressed as percentages of the total radioactivity in the glycoprotein). Neutral sugar, hexosamine and N-acetylneuraminic acid residues of the mucoprotein were each labelled to a similar extent. 4. Similarly, the ratio of the radioactivity in N-glycollylneuraminic acid to that in N-acetylneuraminic acid in the mucoprotein from tissue incubations with [2−14C]-acetate was 1·0:4·0. 5. Both [2−14C]acetate and [2−14C]glucose with whole tissue led to labelling of the N-glycollyl substituent and of the main nonose skeleton of the N-glycollylneuraminic acid. In whole-tissue incubations, [3−14C]pyruvate was also a precursor of radioactive N-glycollylneuraminic acid. 6. Hydroxy[3−14C]-pyruvate and [U−14C]glycine caused labelling of the carbohydrate and peptide residues of the glycoprotein, but did not give rise to labelling in the N-glycollylneuraminic acid residues. 7. With a wide variety of possible N-glycollyl precursors (fructose 6-phosphate, hydroxypyruvate, glycollate and chemically synthesized glycollyl-CoA) biosynthesis of N-glycollylglucosamine was not observed in cell-free preparations.

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