A membrane-bound trehalase from Chironomus riparius larvae: purification and sensitivity to inhibition.
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 microM), deoxynojirimycin (IC(50) = 2.83 +/- 0.34 microM) and castanospermine (IC(50) = 12.7 +/- 1.4 microM). 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.
Forcella
Matilde
M
Department of Environmental Sciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.
Cardona
Francesca
F
Goti
Andrea
A
Parmeggiani
Camilla
C
Cipolla
Laura
L
Gregori
Maria
M
Schirone
Raffaella
R
Fusi
Paola
P
Parenti
Paolo
P
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002683:Chironomidae / Q000201:enzymology* / Q000254:growth & development; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors; D007700:Kinetics; D007814:Larva / Q000737:chemistry / Q000201:enzymology*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008954:Models, Biological; D011758:Pyrroles / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D014198:Trehalase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism*
D000470:Alkaloids; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D011758:Pyrroles; C409108:casuarine; C501633:deoxynojirimycine; D017485:1-Deoxynojirimycin; D006026:Glycoside Hydrolases; D014198:Trehalase; D005944:Glucosamine