Effects of Inhibitors on Haemolymph Phenoloxidase From Rosaceous Branch Borer, Ospheranteria Coerulescens (Coleoptera: Cerambycidae)

Abstract

The rosaceous branch borer, Ospheranteria coerulescens, is an important pest of rosaceous trees. This insect feeds on the twigs and branches of living trees and causes their death. The characterization of the insect phenoloxidase (PO) is of interest when doing comparative investigations, and so that we may be able to understand its biochemical properties. When designing new methods of insect control such as the use of PO inhibitors, an understanding of the biochemical properties is fundamental. In this study, PO from hemolymph of the rosaceous branch borer was purified using ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography. The biochemical properties were characterized using l-dihydroxyphenylalanine (L-DOPA) as the specific substrate. The apparent molecular weights of the three isoforms of PO were determined by SDS-PAGE to be 85.23, 79.45, and 66.06 kDa. Optimal pH for PO activity was pH 8, and the optimal temperature was 45°C. Phenoloxidase lost less than 50% of its relative activity after a 60 min incubation at the optimal temperature. The effects of ions and chemical materials such as K + , Ba 2+ , Zn 2+ and EDTA on PO showed that PO activity was strongly inhibited by Zn 2+ . The Michaelis constant (K m ) and maximum velocity (V max ) were 88.61 mM and 0.14 µmol/min, respectively. The inhibitory effects of kojic acid, 4-hexylresorsinol, and quercetin on PO were determined, and the IC 50s (inhibitory concentration) were estimated as 23.31 for kojic acid, 35.75 for 4-hexylresorcinol, and 60.8 µM for quercetin. The inhibitory potency of kojic acid was 1.54 times higher than that of 4-hexylresorcinol and 2.58 times higher than that of quercetin. Phenoloxidase was effectively inhibited by 4-hexylresorcinol, and the inhibition type was competitive. The inhibition types of PO by kojic acid and quercetin were found to be mixed.

Purification and characterization of phenoloxidase from the hemolymph of healthy and diseasedAntheraea assamensisHelfer (Lepidoptera: Saturniidae): Effects of certain biological components and chemical agents on enzyme activity

“…Zibaee et al () found that Ca 2+ and Cu 2+ increased PO activity, whereas Zn 2+ showed no effect in E. integriceps . Zn 2+ also inhibited PO activity of Ospheranteria coerulescens (Gholami, Ghadamyari, Oliaee, & Ajamhassani, ) and Charybdis japonica (Liu et al, ), a report similar to the present finding. Most intriguingly, Ca 2+ increased PO activity in B. Mori (Ashida et al, ), which did not show much significance in our study.…”

“…The enhancement of PO activity in presence of mono and divalent cations have been studied in Hyalophora cecropia (Anderson, Sun, Boman, & Steiner, ), Schistocerca gregaria , S. bullata (Chase, Raina, Bruno, & Sugumaran, ), O. furcanalis (C. Feng, Song, Lü, & Lu, ) and G. mellonella (Zibaee et al, ). We did not come across a documented study on the role of cobalt (Co 2+ ) ions on PO activity (Gholami et al, ) and this perhaps in the only report on silkworms. It is assumed that metal ions increase or decrease PO activity by modulating the protein structure whereby secondary structures of certain peptides of PO are affected (C. Feng et al, ; Gholami et al, ; Liu et al, ).…”

“…We did not come across a documented study on the role of cobalt (Co 2+ ) ions on PO activity (Gholami et al, ) and this perhaps in the only report on silkworms. It is assumed that metal ions increase or decrease PO activity by modulating the protein structure whereby secondary structures of certain peptides of PO are affected (C. Feng et al, ; Gholami et al, ; Liu et al, ).…”

Phenoloxidase and its zymogen are required for the larval–pupal transition in Bactrocera dorsalis (Diptera: Tephritidae)

“…Divalent cations ions such as Ca 2+ and Cu 2+ significantly increased the enzymatic activity in E. integriceps (Zibaee et al, 2011). However, this enzyme activity also could be strongly inhibited by Zn 2+ in Ospheranteria coerulescens (Gholami et al, 2013). These findings all suggest that PO may be a metalloprotein.…”

Biological and Histological Effects of Certain Insecticides on Spodoptera littoralis (Bosid)

“…The bio-chemical controlling for some insect-pests is effected with insecticides for example synthetic-insectgrowth-regulator (IGRs), especially; for essential agent action on insect-developments and insect-growthing. Insect-growth-regulator "IGRs" disruption cause regulation hormone association with insect-metamorphosis can ultimately cause death of insect (Gholami et al 2013). Emamectin benzoate (1.9% EC) (Methylamin :Avermactin) belongs to A vermactin group of chemicals which block the post synaptic potential of neuro muscular junction leading to paralysis and finally to the death.…”