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.
el Moudni
B
B
Laboratoire de Parasitologie et Mycologie Médicale, Centre Hospitalier Universitaire, La Milétrie, Poitiers, France.
Rodier
M H
MH
Jacquemin
J L
JL
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008970:Molecular Weight; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D005456:Fluorescent Dyes; C025158:4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside; D006923:Hymecromone; D000118:Acetylglucosaminidase; D000117:Acetylglucosamine