The kinetic parameters of partially purified phenoloxidase （PO, EC. 1.14.18.1） from the 5th instar larvae of Pieris rapae （Lepidoptera） were determined, using L-3, 4- dihydroxyphenylalanine （L-DOPA） as substrate...The kinetic parameters of partially purified phenoloxidase （PO, EC. 1.14.18.1） from the 5th instar larvae of Pieris rapae （Lepidoptera） were determined, using L-3, 4- dihydroxyphenylalanine （L-DOPA） as substrate. The optimal pH and temperature of the enzyme for the oxidation of L-DOPA were determined to be at pH 7.0 and at 42℃, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37℃. At pH 6.8 and 37℃, the Michaelis constant （Kin） and maximal velocity （V） of the enzyme for the oxidation of L-DOPA were determined to be 0.80 mmol/L and 1.84 μmol/ L/min, respectively. Tetra-hexylresorcinol and 4-dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC50 of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47μmol/L, respectively.展开更多
Insecticidal effects of 4-hexylresorcinol, a phenoloxidase （PO） inhibitor, were determined on Hyphantria cunea （Drury） under laboratory conditions. The LC50 for the 15- d-old larvae was estimated to be 2.95 g/L af...Insecticidal effects of 4-hexylresorcinol, a phenoloxidase （PO） inhibitor, were determined on Hyphantria cunea （Drury） under laboratory conditions. The LC50 for the 15- d-old larvae was estimated to be 2.95 g/L after 96 h exposure. The activities of glutathione S-transferase （GST） and PO showed a decrease in larvae treated with 4-hexylresorcinol, and the IC50 of GST and PO were estimated to be 0.8 and 0.43 g/L, respectively, 24 h after treatment. The PO from the hemolymph of fall webworm was purified by ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography, and then enzymatic characteristics and the mechanism of inhibition were determined using L-dihydroxyphenylalanine （L-DOPA） as the substrate. The purified PO showed a single band on SDS-PAGE with a molecular weight of about 70 kDa. The optimum pH for PO activity was observed at pH 7.0, optimum temperature was found to be 45 ℃, and PO activity was strongly inhibited by Zn2＋. IC50 values were estimated to be 8.2, 19.14, and 24.04/zmol/L for 4-hexylresorsinol, kojic acid, and quercetin, respectively. The inhibitory potencies （i.e., 150 of each compound/Is0 of 4-hexylresorcinol） ofkojic acid and quercetin on H. cunea PO were estimated to be 1.87 and 2.89, respectively. 4-hexylresorcinol was determined to be a competitive inhibitor, and kojic acid and quercetin were determined to be mixed inhibitors. PO is one of the most important enzymes in an insect＇s immune system, and the use ofPO inhibitors seems to be a promising approach for pest control due to their potential safety for humans.展开更多
基金Acknowledgments The present investigation was supported in part by grant N0. 30270887, 30571237 of the National Natural Science Foundation of China for W. C. Luo.
文摘The kinetic parameters of partially purified phenoloxidase （PO, EC. 1.14.18.1） from the 5th instar larvae of Pieris rapae （Lepidoptera） were determined, using L-3, 4- dihydroxyphenylalanine （L-DOPA） as substrate. The optimal pH and temperature of the enzyme for the oxidation of L-DOPA were determined to be at pH 7.0 and at 42℃, respectively. The enzyme was stable between pH 6.5 and 7.4 and at temperatures lower than 37℃. At pH 6.8 and 37℃, the Michaelis constant （Kin） and maximal velocity （V） of the enzyme for the oxidation of L-DOPA were determined to be 0.80 mmol/L and 1.84 μmol/ L/min, respectively. Tetra-hexylresorcinol and 4-dodecylresorcinol effectively inhibited activity of phenoloxidase and this inhibition was reversible and competitive, with the IC50 of 1.50 and 1.12 μmol/L, respectively. The inhibition constants were estimated to be 0.50 and 0.47μmol/L, respectively.
文摘Insecticidal effects of 4-hexylresorcinol, a phenoloxidase （PO） inhibitor, were determined on Hyphantria cunea （Drury） under laboratory conditions. The LC50 for the 15- d-old larvae was estimated to be 2.95 g/L after 96 h exposure. The activities of glutathione S-transferase （GST） and PO showed a decrease in larvae treated with 4-hexylresorcinol, and the IC50 of GST and PO were estimated to be 0.8 and 0.43 g/L, respectively, 24 h after treatment. The PO from the hemolymph of fall webworm was purified by ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography, and then enzymatic characteristics and the mechanism of inhibition were determined using L-dihydroxyphenylalanine （L-DOPA） as the substrate. The purified PO showed a single band on SDS-PAGE with a molecular weight of about 70 kDa. The optimum pH for PO activity was observed at pH 7.0, optimum temperature was found to be 45 ℃, and PO activity was strongly inhibited by Zn2＋. IC50 values were estimated to be 8.2, 19.14, and 24.04/zmol/L for 4-hexylresorsinol, kojic acid, and quercetin, respectively. The inhibitory potencies （i.e., 150 of each compound/Is0 of 4-hexylresorcinol） ofkojic acid and quercetin on H. cunea PO were estimated to be 1.87 and 2.89, respectively. 4-hexylresorcinol was determined to be a competitive inhibitor, and kojic acid and quercetin were determined to be mixed inhibitors. PO is one of the most important enzymes in an insect＇s immune system, and the use ofPO inhibitors seems to be a promising approach for pest control due to their potential safety for humans.
