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The biosynthesis of N-glycoloylneuraminic acid in fractionated porcine submandibular glands was investigated. The following substrates: 3HN-acetylmannosamine, free 14CN-acetylneuraminic acid, CMP-14CN-acetylneuraminic acid, 14CN-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 14CN-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-14CN-acetylneuraminic acid, was hydroxylated. The product was identified as CMP-14CN-glycoloylneuraminic acid by (i) co-chromatography with non-radioactive CMP-N-glycoloylneuraminic acid standard, (ii) acid hydrolysis to free 14CN-glycoloylneuraminic acid, (iii) alkaline hydrolysis to yield N-glycoloylneuraminic acid and 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid and (iv) transfer of 14CN-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.
Shaw et al. (Fri,) studied this question.