Purification and characterization of a phenoloxidase in the hemocytes ofEphestia kuehniellaZeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors

Abstract

A phenoloxidase was extracted and purified from hemocytes of Ephestia kuehniella by using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose fast flow chromatographies. At the final stage of purification, a protein was purified by molecular mass of 78.5 kDa, specific activity of decreased activity of the purified PO but Ca 2+ and Cu 2+ increased the enzymatic activity. EDTA (General chelating agent), DTC (Copper chelating agent) and EGTA (Calcium chelating agent) significantly decreased PO activity but TTHA (Magnesium chelating agent) showed no statistically significant effects. Kinetic parameters of the purified enzyme showed the highest V max when L-DOPA was used as substrate but no significant differences were observed in case of K m for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC 50 of 96.41 and 38.59 mg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC 50 concentrations of the two IGRs revealed the increase of K m value versus control and competitive inhibition. Finally, column chromatography of hemolymph revealed peak III showing endogenous inhibitors of phenoloxidase by molecular weight of 27.3 that showed competitive inhibition on the PO.

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

“…The effect of external agents as potent activators of PO has been reported in a large number of insect species (Delkash‐Roudsari et al, ). The activation of PPO to PO has been achieved through agents like SDS (Fan et al, ), CPC (Goudru et al, ), or chymotrypsin (Kopacek et al, ).…”

“…The desalted Bio‐Gel P‐100 (Bio‐Rad Laboratories, Hercules, CA) portions of HLS‐H and HLS‐D were loaded separately onto a prepacked Sephacryl® S‐100 HiPrep gel filtration column (GE Healthcare Bio‐Sciences AB, Uppsala, Sweden) (1.6 cm × 60 cm) connected to Bio‐Rad NGC™. The column was equilibrated with 20 mM Tris‐Cl (pH 7.0) containing 0.05% (vol/vol) Triton X‐100 (Delkash‐Roudsari, Zibaee, & Bigham, ). Protein fractions were eluted in the same buffer at a flow rate of 0.5 ml/min and 22 psi.…”

“…The molecular mass of PO was estimated using standard marker (medium range, 14.3–97.4 kDa; Genie™, Bengaluru, India) having the standards: phosphorylase‐b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soybean trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa). After SDS‐PAGE, proteins were stained with 0.2% Coomassie brilliant blue R‐250 and destained in methanol and acetic acid (Delkash‐Roudsari et al, ).…”

“…The reaction mixtures comprised of 50 µl of 20 mM Tris‐Cl buffer (pH 7.2), 10 µl of 10% CPC, 20 µl of 6 mM L ‐DOPA, 10 µl of cations in the mentioned concentrations, and 10 µl crude and purified PO. The mixtures were incubated for 10 min and absorbance was read at 495 nm (Delkash‐Roudsari et al, ). The effect of five copper chelators namely, benzoic acid (GRM1326; Himedia), kojic acid (501304; Merck), sodium azide (26628228; Merck), thiourea (GRM611; Himedia), and dithiothreitol (RM525; Himedia), at concentrations of 1, 5, and 10 mM were tested on 20 µl of crude and purified PO extracts.…”

“…The reaction mixture comprised of 940 µl of 20 mM Tris‐Cl (pH 7.2) buffer, 20 µl of 6 mM L ‐DOPA, 10 µl of mentioned concentrations of chelators, and 20 µl of crude and purified PO extracts (Goudru et al, ). Rest of the procedure followed is similar to the methodology mentioned above (Delkash‐Roudsari et al, ). The effect of chemical inhibitors of PO namely, phenylthiourea (GRM6157; Himedia), diethyldithiocarbamate (20624253; Merck), carbendazim (10605217; Merck), N ‐bromosuccinimide (128085; Merck), and N , N , N ′, N ′‐tetraacetic acid (EGTA; 85233198; Merck) were also tested at 1 mM concentration (Delkash‐Roudsari et al, ).…”