The Partial Purification of Two β-N-Acetyl-D-hexosaminidases from Porcine Kidney
Two distinct fractions showing both β-N-acetyl-D-glucosaminidase (EC 3.2.1.30) and β-N-acetyl-D-galactosaminidase activity were isolated and purified from pig kidney. These preparations, which were designated A and B, were not stable during gel chromatography or prolonged dialysis. Final purifications of 600-fold for enzyme A and 440-fold for enzyme B were obtained.Gel electrophoresis and ultracentrifugation studies indicated heterogeneity in both preparations. The amino acid compositions of both preparations were very similar. Ultracentrifugation studies suggested the formation of subunits in the presence of 5 M guanidine–HCl and 1 mM dithiothreitol.A study of the enzymatic properties also showed great similarities between the two enzyme forms. Both enzymes had identical Michaelis–Menten constants of 1.88 mM for p-nitrophenyl-β-N-acetyl-D-glucosaminide and 0.38 mM for p-nitrophenyl-β-N-acetyl-D-galactosaminide. Although bovine serum albumin enhanced the activity of the enzymes it did not change the Km values. The pH-rate profiles of both enzymes with the substrate p-nitrophenyl-β-N-acetyl-D-glucosaminide showed two peaks. When p-nitrophenyl-β-N-acetyl-D-galactosaminide was used as substrate, only one peak was observed in the pH–rate profiles. However, in this case a distinct shoulder could be detected in these peaks. Heating at 50° destroyed the activities of both forms of the enzyme rapidly, but addition of bovine serum albumin protected against heat inactivation.