| Phenoloxidase (PO, EC.1.14.18.1), is a copper-containing enzyme widely distributed in plants, microorganisms and animals. This multifunctional enzyme catalyzes two distinct reactions, the hydroxylation of monophenol to o-diphenol (monophenolase activity) and the conversion of o-diphenol to the corresponding o-quinone (diphenolase activity). It is one of the key enzymes in the development process of insects, the enzyme possesses an important function in metamorphism developing and immunity system. Currently, many studies focused on this field in order to screen, design and synthesis PO inhibitors for the importance theory of PO inhibitors and its bright future.In the present paper, the kinetic properties of phenoloxidase from Lymantria dispar , Sidemia depravata , two kinds of Lepidoptera insect, Gastrolina depressa and Holotrichia oblita, two kinds of Coleoptera insects, were determined after the enzyme was partially purified by different staturated (NH4)2SO4 and Sephadex G-100 gel. The effects of some metal ions and several organic solvents on the activity of the PO were also studied. The inhibitory effects on the PO from Lymantria dispar activity by quercetin and 4-dodecylresorcinol were determined, and the mechanism of the two inhibitors were discussed also. The inhibitory effects of tetra-hexylresorcinol and kojic acids on the monophenolase and diphenolase activities of PO were also studied in the present paper. The inhibitory effects on the PO from Gastrolina depressa and Sidemia depravata activity by corbic acid and cysteine were determined, and the mechanism of the two inhibitors were discussed also. The results could be summarized as follows:1. The kinetic properties of phenoloxidase from Lymantria dispar, Sidemia depravata, two kinds of Lepidoptera insects, and Gastrolina depressa , Holotrichia oblita, two kinds of Coleoptera insects, were determined after the enzyme was partially purified by different staturated (NH4)2SO4. The results showed that the optimum pH of phenoloxidase from Lymantria dispar, Gastrolina depressa was 6.5, 7.0 and the optimum temperature was 35℃and 25℃, respectively. The kinetic parameter for the oxidation of L-DOPA, catechol and pyrogallol by PO from Lymantria dispar were determined, the Km were 3.19, 10.74 and 19.26 mmol·L-1, respectively. The kinetic parameter for the oxidation of L-DOPA and catechol by PO from Sidemia depravata were determined too, the Km were 37.49 and 1.44 mmol·L-1, respectively. The results also showed that the optimum pH of phenoloxidase from Gastrolina depressa and Holotrichia oblita were 7.5, 7.0, respectively and the optimum temperature was consistent 40℃. The kinetic parameter for the oxidation of L-DOPA and catechol by PO from Gastrolina depressa and Holotrichia oblita were determined too, the Km were 15.01 and 9.17 mmol·L-1, 3.02 and 61.25 mmol·L-1, respectively.2. The effect of some metal ions on the PO activity was studied. The results showed that alkali metal ions K+ and Na+ had no significantly influences on PO from Gastrolina depressa, Sidemia depravata and Holotrichia oblita activity, however K+ showed a slight inhibition role to PO from Lymantria dispar. Alkali-earth metal ion Mg2+ showed inhibition role to PO from Lymantria dispar and promotion role to PO from Gastrolina depressa and Sidemia depravata. Mg2+enhanced the enzyme activity when it was at the low concentration, but the activity was inhibited by Mg2+ when the concentration went over to 0.40 mmol·L-1. PO from Gastrolina depressa and Sidemia depravata activity were inhibited by Ba2+, but this ion had no significantly influences on PO from Holotrichia oblita activity. The activity of PO from Lymantria dispar, Sidemia depravata and Holotrichia oblita activation and inhibtion were observed at low and high concentration of Cu2+, but Cu2+ showed inhibition role to the PO from Gastrolina depressa activity. Zn2+ showed activation and inhibition role to the activity of PO from Sidemia depravata, Gastrolina depressa, respectively. Zn2+ played the same role as Cu2+ to the activity of PO from Lymantria dispar and Holotrichia oblita. Mn2+ showed no significantly influences on PO from Sidemia depravata. The activity of PO from Holotrichia oblita activation and inhibtion were observed at low and high concentration of Mn2+.3. The effect of some organic solvents on the PO activity was studied. The results showed that ethyl ether showed remarkable inhibition role to the activity of PO from Sidemia depravata and Holotrichia oblita. Ethanol and glycerol also showed remarkable inhibition role to the activity of PO from Gastrolina depressa and Holotrichia oblita, respectively. Ethanol can actived the activity of PO from Sidemia depravata slightly. The activity of PO from Lymantria dispar and Holotrichia oblita can be inhibited by acetone, however, acetone had neither inhibition nor activation to the activity of PO from Sidemia depravata.4. Quercetin and 4-dodecylresorcino showed remarkable inhibition role to the activity of PO from Lymantria dispar. The two inhibitors concentrations leading to 50% (IC50) activity lost were estimated to be 0.076 mmol·L-1 and 0.372 mmol·L-1 , respectively, using catechol as substrate. Both quercetin and 4-dodecylresorcino were reversible competitive inhibitors and the inhibitory constants (Ki) were determined to be 31.71 mmol·L-1 and 0.192 mmol·L-1 repectively. The inhibitory effects of tetra-hexylresorcinol and kojic acids on the phenoloxidase from Lymantria dispar were also stuided, using L-tyrosine or catechol as substrate. The results showed that the IC50 were estimated to be 0.00041 mmol/L for monophenolase activity and 0.00035 mmol/L for diphenolase, respectively. Tetra-hexylresorcinol extended the lagtime of the enzyme for oxidation of L-tyrosine. The lagtime extended from 181 s to 253s by 4-hexylresorcinol with 0.0002 mmol/L, and the lagtime extended from 181 s to 372s by the natural source compound with 0.0005 mmol/L. The results of inhibition kinetics analyzed by Lineweaver-Burk plots indicated that 4-hexylresorcinol was a competitive inhibitor for the oxidation of catechol and the inhibition constant was determined to be 0.00015 mmol/L.The results showed that the IC50 were estimated to be 0.06 mmol/L for monophenolase activity and 0.92 mmol/L for diphenolase, respectively. Kojic acids extended the lagtime of the enzyme for oxidation of L-tyrosine. The lagtime extended from 306 s to 702s by the kojic acids with 0.1 mmol/L, and the lagtime extended from 306 s to 900s by the compound with 0.15 mmol/L. The results of inhibition kinetics analyzed by Lineweaver-Burk plots indicated that kojic acids was a competitive inhibitor for the oxidation of catechol, and the inhibition constant was determined to be 0.51 mmol/L.PO from Gastrolina depressa was insensitive to ascorbic acid. The PO activity decreased with ascorbic acid showing a 27% most reduction as compared with no inhibitor in and scorbic acid was a reversible competitive inhibitors to this PO . PO from Sidemia depravata was sensitive to ascorbic acid which was a reversible competitive inhibitors for this PO and the Ki were estimated to be 0.0768 mmol·L-1. PO from Gastrolina depressa was sensitive to cystein and this inhibitor which was an irreversible competitive inhibitors and the inhibitory constants (Ki) were determined to be 0.40 mmol·L-1 can almost inhibited the activity completely. PO from Sidemia depravata was sensitive to cystein which was a reversible competitive inhibitors for this PO and the Ki were estimated to be 0.0742 mmol·L-1.
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