A preparation of a membrane-bound trehalase from 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. Initial rate of trehalase activity followed Henri-Michaelis-Menten kinetics with a Km 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 (IC50 = 0.25 ± 0.03 μM), deoxynojirimycin (IC50 = 2.83 ± 0.34 μM), and castanospermine (IC50 = 12.7 ± 1.4 μM). 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 pest having trehalase as biochemical target can be developed.

A membrane-bound trehalase from chironomus riparius larvae: purification and sensitivity to inhibition / M. FORCELLA; F. CARDONA; A. GOTI; C. PARMEGGIANI; L. CIPOLLA; M. GREGORI; R. SCHIRONE; P. FUSI; P. PARENTI. - In: GLYCOBIOLOGY. - ISSN 0959-6658. - STAMPA. - 20:(2010), pp. 1186-1195. [10.1093/glycob/cwq087]

A membrane-bound trehalase from chironomus riparius larvae: purification and sensitivity to inhibition

CARDONA, FRANCESCA;GOTI, ANDREA;PARMEGGIANI, CAMILLA;
2010

Abstract

A preparation of a membrane-bound trehalase from 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. Initial rate of trehalase activity followed Henri-Michaelis-Menten kinetics with a Km 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 (IC50 = 0.25 ± 0.03 μM), deoxynojirimycin (IC50 = 2.83 ± 0.34 μM), and castanospermine (IC50 = 12.7 ± 1.4 μM). 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 pest having trehalase as biochemical target can be developed.
2010
20
1186
1195
M. FORCELLA; F. CARDONA; A. GOTI; C. PARMEGGIANI; L. CIPOLLA; M. GREGORI; R. SCHIRONE; P. FUSI; P. PARENTI
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Utilizza questo identificatore per citare o creare un link a questa risorsa: https://hdl.handle.net/2158/393790
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