title: The biosynthesis of N-glycoloylneuraminic acid occurs by hydroxylation of the CMP-glycoside of N-acetylneuraminic acid.
abstract: The biosynthesis of N-glycoloylneuraminic acid in fractionated porcine submandibular glands was investigated. The following substrates: [3H]N-acetylmannosamine, free [14C]N-acetylneuraminic acid, CMP-[14C]N-acetylneuraminic acid, [14C]N-acetylneuraminic acid linked alpha(2----3) to galactose residues, or alpha(2----6) to Gal-beta(1----4)-GlcNAc residues of porcine submandibular mucin and [14C]N-acetylneuraminic acid linked alpha(2----6) to GalNAc residues of ovine submandibular gland mucin were incubated, in the presence of cofactors, with the soluble protein, heavy membrane and microsomal fractions of porcine submandibular glands. Radio thin-layer chromatographic analysis revealed that only one substrate, CMP-[14C]N-acetylneuraminic acid, was hydroxylated. The product was identified as CMP-[14C]N-glycoloylneuraminic acid by (i) co-chromatography with non-radioactive CMP-N-glycoloylneuraminic acid standard, (ii) acid hydrolysis to free [14C]N-glycoloylneuraminic acid, (iii) alkaline hydrolysis to yield N-glycoloylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid and (iv) transfer of [14C]N-glycoloylneuraminic acid to asialo-fetuin by sialyltransferase. 85% of CMP-N-acetylneuraminic acid hydroxylase activity was present in the soluble protein fraction, with small amounts of activity in the two particulate fractions. The CMP-N-acetylneuraminic acid hydroxylase in the soluble protein fraction had an absolute requirement for Fe2+ ions and a reducing cofactor. NADPH and NADH were by far the most effective cofactors, smaller amounts of hydroxylation could, however, be supported by ascorbic acid and 6,7-dimethyl-5,6,7,8-tetrahydrobiopterin.
authors.affiliation: Biochemisches Institut der Universität zu Kiel.
authors.forename: L
authors.initials: L
authors.lastname: Shaw
authors.forename: R
authors.initials: R
authors.lastname: Schauer
mesh_terms: D000818:Animals; D002250:Carbon Radioisotopes; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System; D006595:Hexosamines / Q000378:metabolism; D006900:Hydroxylation; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009077:Mucins / Q000378:metabolism; D019158:N-Acetylneuraminic Acid; D009438:Neuraminic Acids / Q000096:biosynthesis*; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism; D013250:Steroid Hydroxylases; D013347:Subcellular Fractions / Q000201:enzymology; D013363:Submandibular Gland / Q000201:enzymology*; D013552:Swine; D014316:Tritium
chemical_list: D002250:Carbon Radioisotopes; D006595:Hexosamines; D009077:Mucins; D009438:Neuraminic Acids; D012794:Sialic Acids; D014316:Tritium; C032592:N-glycolylneuraminic acid; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C074383:CMPacetylneuraminate monooxygenase; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine