Detection of CMP-N-acetylneuraminic acid hydroxylase activity in fractionated mouse liver.
The finding that N-glycoloylneuraminic acid (Neu5Gc) in pig submandibular gland is synthesized by hydroxylation of the sugar nucleotide CMP-Neu5Ac [Shaw & Schauer (1988) Biol. Chem. Hoppe-Seyler 369, 477-486] prompted us to investigate further the biosynthesis of this sialic acid in mouse liver. Free [14C]Neu5Ac, CMP-[14C]Neu5Ac and [14C]Neu5Ac glycosidically bound by Gal alpha 2-3- and Gal alpha 2-6-GlcNAc beta 1-4 linkages to fetuin were employed as potential substrates in experiments with fractionated mouse liver homogenates. The only substrate to be hydroxylated was the CMP-Neu5Ac glycoside. The product of the reaction was identified by chemical and enzymic methods as CMP-Neu5Gc. All of the CMP-Neu5Ac hydroxylase activity was detected in the high-speed supernatant fraction. The hydroxylase required a reduced nicotinamide nucleotide [NAD(P)H] coenzyme and molecular oxygen for activity. Furthermore, the activity of this enzyme was enhanced by exogenously added Fe2+ or Fe3+ ions, all other metal salts tested having a negligible or inhibitory influence. This hydroxylase is therefore tentatively classified as a monooxygenase. The cofactor requirement and CMP-Neu5Ac substrate specificity are identical to those of the enzyme in high-speed supernatants of pig submandibular gland, suggesting that this is a common route of Neu5Gc biosynthesis. The relevance of these results to the regulation of Neu5Gc expression in sialoglycoconjugates is discussed.
Shaw
L
L
Biochemisches Institut, Christian-Albrechts-Universität Kiel, Federal Republic of Germany.
Schauer
R
R
D000818:Animals; D002458:Cell Fractionation; D002855:Chromatography, Thin Layer; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D005290:Ferric Compounds / Q000494:pharmacology; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008807:Mice, Inbred BALB C; D019158:N-Acetylneuraminic Acid; D009243:NAD / Q000494:pharmacology; D009438:Neuraminic Acids / Q000378:metabolism* / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012794:Sialic Acids / Q000378:metabolism; D013379:Substrate Specificity
D005290:Ferric Compounds; D009438:Neuraminic Acids; D012794:Sialic Acids; D009243:NAD; C032592:N-glycolylneuraminic acid; C024823:ferric sulfate; C060603:cytidine monophosphate-N-glycoloylneuraminic acid; D010727:Phosphoric Diester Hydrolases; C010265:CMP-N-acylneuraminate phosphodiesterase; D003568:Cytidine Monophosphate; D019158:N-Acetylneuraminic Acid