A membrane-bound trehalase from Chironomus riparius larvae: purification and sensitivity to inhibition
Year: 2010
Authors: Forcella M., Cardona F., Goti A., Parmeggiani C., Cipolla L., Gregori M., Schirone R., Fusi P., Parenti P.
Autors Affiliation: Department of Environmental Sciences, and Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy, and Department of Chemistry “U. Schiff”, Laboratory of Design, Synthesis and Study of Biologically Active Heterocycles (HeteroBioLab), University of Florence, Via della Lastruccia 13, 50019
Florence, Italy
Abstract: A preparation of a membrane-bound trehalase from the larvae of the midge Chironomus riparius (Diptera: Chironomidae) was obtained by detergent solubilization, ion-exchange chromatography and concanavalin A affinity chromatography. Trehalase was purified 1080-fold to a specific activity of 75 U mg(-1). The initial rate of trehalase activity followed Henri-Michaelis-Menten kinetics with a K(m) of 0.48 +/- 0.04 mM. Catalytic efficiency was maximal at pH 6.5. The activity was highly inhibited by mono- and bicyclic iminosugar alkaloids such as (in order of potency) casuarine (IC(50) = 0.25 +/- 0.03 mu M), deoxynojirimycin (IC(50) = 2.83 +/- 0.34 mu M) and castanospermine (IC(50) = 12.7 +/- 1.4 mu M). Increasing substrate concentration reduced the inhibition. However, in the presence of deoxynojirimycin, Lineweaver-Burk plots were curvilinear upward. Linear plots were obtained with porcine trehalase. Here, we propose that deoxynojirimycin inhibits the activity of trehalase from C. riparius according to a ligand exclusion model. Inhibition was further characterized by measuring enzyme activity in the presence of a series of casuarine and deoxynojirimycin derivatives. For comparison, inhibition studies were also performed with porcine trehalase. Results indicate substantial differences between midge trehalase and mammalian trehalase suggesting that, in principle, inhibitors against insect pests having trehalase as biochemical targets can be developed.
Journal/Review: GLYCOBIOLOGY
Volume: 20 (9) Pages from: 1186 to: 1195
More Information: We thank Carla Smeraldi for proofreading the manuscript in its final form. This work was supported by a grant from the University of Milano-Bicocca (FAR 2008). We also thank Consorzio Interuniversitario Nazionale \”Metodologie e Processi Innovativi di Sintesi\” (C.I.N.M.P.I.S.), MIUR (PRIN 2008) and Ente Cassa di Risparmio di Firenze, Italy, for financial support.DOI: 10.1093/glycob/cwq087ImpactFactor: 3.791Citations: 36data from “WEB OF SCIENCE” (of Thomson Reuters) are update at: 2024-11-10References taken from IsiWeb of Knowledge: (subscribers only)Connecting to view paper tab on IsiWeb: Click hereConnecting to view citations from IsiWeb: Click here
