Characterization of Chitobiase from Daphnia magna and Its Relation to Chitin Flux

Abstract

Chitobiase (N-acetyl-β-D-glucosaminidase) activity has been measured and partially characterized in Daphnia magna. The pH optimum is approximately 5.5, and the temperature optimum is approximately 45°dg-5° dgC, as determined from crude extract. The enzyme remains stable even after a 2-h incubation at 20° or 40°C. The Km determined for methylumbelliferyl-N-acetyl-β-D-glucosaminide and p-nitrophenyl-N-acetyl-β-D-glucosaminide is 61.5 μM and 359.6 μM, respectively. Activation energy is significantly less for lower temperatures (5°-18°C) than for higher temperatures (18°-40°C). Investigation of the effect of acclimation temperature on chitobiase activities reveals no significant differences in activities between 10° and 18°C for populations raised at different temperatures. A similar pattern in the Q10's was also observed. There was no evidence of any isozyme effect for populations raised at 6°, 10°, 18°, or 25°C Attempts to protect the enzyme with various osmotic solutions and the results of differential centrifugation suggest that at least some of the enzyme is packaged in vesicles and/or lysosomes. Maximal chitobiase activity of asynchronous populations fed ad lib. overestimates the flux of N-acetyl-β-D-glucosaminide (NAG) from the old to the new cuticle by approximately 100×. Chitobiase activity modulates significantly over the molt cycle in Daphnia, with a fivefold increase in activity 6-0 h before molt. This suggests that chitobiase activity may provide an index of molt rate in asynchronously molting populations.