Separation of N ‐Acetyl‐α‐Glucosaminidase and N ‐Acetyl‐α‐Galactosaminidase from Ox Spleen

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.