KINETIC-PARAMETERS OF N-ACETYLGLUCOSAMINIDASE IN ADULT FEMALE NIPPOSTRONGYLUS-BRASILIENSIS BY A QUANTITATIVE COLORIMETRIC MICROMETHOD

Chitin, the 1,4-beta-linked homopolymer of N-acetylglucosamine, is a c omponent of Nematode eggshell. Chitin is hydrolyzed to its constituent monomer by the binary chitinase enzyme system. Chitinase (EC 3.2.1.14 ) hydrolyses first the polymer to N,N'-diacetylchitobiose which is the n split to N-acetylglucosamine by N-acetylglucosaminidase (EC 3.2.1.30 ). N-acetylglucosaminidase is also able to release N-acetylglucosamine monomers directly from chitin. This study reports the identification of N-acetylglucosaminidase in adult female Nippostrongylus brasiliensi s. A quantitative colorimetric micromethod has been developed to detec t N-acetylglucosaminidase activity from N. brasiliensis using p-nitrop henyl-N-acetylglucosaminide as substrate. The assay conditions were op timized as follows: 50 mu l of protein extract were incubated at 37 de grees C for 2 h in the presence of 50 mu l of 19 mM substrate solution and 50 mu l of 0.2 M potassium phosphate buffer pH 6.0. The reaction was stopped by the addition of 50 mu l 0.1 N NaOH and the absorbance w as measured on a Multiskan MS spectrophotometer at 405 nm within 30 mi n. after the reaction was stopped. The optimal pH was 6.0. Specific ac tivity of protein extract was found at 94.4 +/- 5.6 pmol/min/mg protei n (n = 4). V-max app. and K-m app. for p-nitrophenyl-N-acetylglucosami nide were respectively 280 +/- 11 pmol/min/mg protein (n = 3) and 1.8 +/- 0.2 mM (n = 3). This test can now be proposed as a tool for screen ing anthelmintic compounds in the field of new inhibitors of chitin me tabolism.