Separation of N-acetyl-alpha-glucosaminidase and N-acetyl-alpha-galactosaminidase from ox spleen. Cleavage of the O-glycosidic linkage between carbohydrate and polypeptide in ovine and bovine submaxillary glycoprotein by N-acetyl-alpha-galactosaminidase.

Abstract

1 Two specific N-acetyl-α-hexosaminidases were detected in bovine spleen homogenates, purified by ammonium sulphate precipitation, separated by gel filtration and identified as N-acetyl-α-glucosaminidase and N-acetyl-α-galactosaminidase respectively. In contrast to the established N-acetyl-β-hexosaminidase which splits phenyl-β-d-glycosides of both N-acetyl-glucosamine and N-acetylgalactosamine, the two N-acetyl-α-hexosaminidases exhibits an absolute specificity towards the hexosamine moiety of their substrates. N-Acetyl-α-galactosaminidase acts on phenyl-N-acetyl-α-d-galactosaminide and not on the corresponding glucosaminide. The reverse holds for N-acetyl-α-glucosaminidase. 2 In the cell-free supernatant of a bovine spleen homogenate N-acetyl-α-galactosaminidase is present with a specific activity about 50-fold of that of the corresponding glucosaminidase, using the appropriate phenyl-α-d-hexosaminides as substrate. 3 Ox spleen N-acetyl-α-galactosaminidase was found to split both phenyl-N-acetyl-α-d-galactosaminide and the O-glycosidic linkage between terminal N-acetylgalactosamine and peptide-bonded serine (threonine) in ovine and bovine submaxillary glycoproteins. The identity of bovine O-seryl-N-acetylgalactosaminide glycosidase and N-acetyl-α-galactosaminidase is clearly demonstrated by the presence of these enzymatic activities in a single chromatographic peak even after re-chromatography. 4 One substrate of natural origin for N-acetyl-α-glucosaminidase was found in UDP-N-acetylglucosamine.