Purification and characterization of N-acetylglucosaminidase from Trypanosoma cruzi.

N-Acetyl-beta-D-glucosaminidase activity was recovered in cell-free extracts of Trypanosoma cruzi epimastigotes. This enzyme was identified on the basis of its ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-beta-D-glucosaminide. This activity was purified to apparent homogeneity by anion exchange and molecular sieve high-performance liquid chromatography. It eluted at a native molecular weight of approximately 48,000 Da and migrated as a single band upon reducing or nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH of the activity was around pH 5.4 and the enzyme gave a single peak of activity on a chromatofocusing column with an isoelectric point of 4.2. The enzyme hydrolyzed 4-methylumbelliferyl-GlcNAc, suggesting that it should be characterized as a N-acetyl-beta-D glucosaminidase, with a K(m) value of 1.5 mM from Lineweaver-Burk plots. Many inhibitors as potential enzyme effectors were investigated.