Substrate specificity of D-galactose oxidase. Evidence for the oxidation of internally linked galactosyl residues of Helix pomatia galactogen.
Linkage analysis of the carbohydrate portion of glycoproteins and glycolipids is widespread. Sequential treatment with D-galactose oxidase and tritiated borohydride is a standard method for incorporation of radioactive marker into what has been assumed to be exclusively terminal residues of D-galactose or N-acetyl-D-galactosamine. The data presented here establishes the ability of D-galactose oxidase to act upon a specific subterminal D-galactosyl residue, [----2)-D-Gal(1----], as well as upon terminal nonreducing galactosyl residues. Helix pomatia galactogen, a high molecular weight galactose homopolymer, was sequentially treated with D-galactose oxidase and tritiated borohydride. The 3H-galactogen was recovered and analyzed to determine which galactosyl units carried radioactive label. After complete methylation and then acid hydrolysis of 3H-galactogen, its partially methylated galactosyl components were reduced and acetylated for identification by gas chromatography and mass spectroscopy. Radioactivity was located by collection of effluent fractions during gas chromatography. The only subterminal residue to be labeled was the 2-linked D-galactose, although another with a free oxidizable 6-carbon was present, 3-linked D-galactose, [----3)-D-Gal(1----]. Linkage analysis of internal radiolabeled galactosyl residues could be used to detect changes in saccharide structure during cellular events.
D000818:Animals; D002849:Chromatography, Gas; D002855:Chromatography, Thin Layer; D005685:Galactans; D005692:Galactose Oxidase / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006372:Helix, Snails; D007700:Kinetics; D008745:Methylation; D013379:Substrate Specificity; D014316:Tritium
D005685:Galactans; D014316:Tritium; C007812:galactogen; D005692:Galactose Oxidase