Phenoloxidase Inhibitors Research Articles

Nucleoprotein of a Rice Rhabdovirus Serves as the Effector to Attenuate Hemolymph Melanization and Facilitate Viral Persistent Propagation in its Leafhopper Vector.

Melanization in the hemolymph of arthropods is a conserved defense strategy against infection by invading pathogens. Numerous plant viruses are persistently transmitted by insect vectors, and must overcome hemolymph melanization. Here, we determine that the plant rhabdovirus rice stripe mosaic virus (RSMV) has evolved to evade the antiviral melanization response in the hemolymph in leafhopepr vectors. After virions enter vector hemolymph cells, viral nucleoprotein N is initially synthesized and directly interacts with prophenoloxidase (PPO), a core component of the melanization pathway and this process strongly activates the expression of PPO. Furthermore, such interaction could effectively inhibit the proteolytic cleavage of the zymogen PPO to active phenoloxidase (PO), finally suppressing hemolymph melanization. The knockdown of PPO expression or treatment with the PO inhibitor also suppresses hemolymph melanization and causes viral excessive accumulation, finally causing a high insect mortality rate. Consistent with this function, microinjection of N into leafhopper vectors attenuates melanization and promotes viral infection. These findings demonstrate that RSMV N serves as the effector to attenuate hemolymph melanization and facilitate viral persistent propagation in its insect vector. Our findings provide the insights in the understanding of ongoing arms race of insect immunity defense and viral counter-defense.

Hemocytes Lysate Supernatant Derived Phenoloxidase Activity in the Hemolymph of Giant Freshwater Prawn Macrobrachium rosenbergii (De Man, 1879)

The aim of the present study attempted to find the cellular immune response in Macrobrachium rosenbergii by melanization reaction produced by hemocytes lysate supernatant (HLS) phenoloxidase (PO) activity. The substrate affinity of the PO enzyme was determined using different phenolic substrates and it was found that the diphenols and polyphenol were oxidized. Hence, the enzyme was characterized as a catechol oxidase type of PO and 3,4-dihydroxy-DL-phenylalanine (DL-DOPA) showed the highest substrate affinity to the HLS. The optimal enzyme activity was observed at 5 mM DL-DOPA in 10 mM Tris-HCl buffer at a pH of 7.5 at 25° C for 20 min and absorbance at 490 nm. Kinetic characteristics of HLS from the prawn were determined. Determination of optimal conditions of PO activity in the HLS has also been attempted. These results depicted that in the presence of PO and peroxidase inhibitors, phenylthiourea (PTU) and tropolone respectively have a decreased HLS PO activity. The determination of PO activity was also highly activated by trypsin, sodium dodecyl sulphate (6 mg.ml-1) and laminarin (4 mg.ml-1) enzyme expression. We also identified the chemicals causing in vitro inhibition or activation of the enzyme as an HLS of the freshwater prawn having a potent PO activity.

Defensive responses of Erianthus arundinaceus against sugarcane shoot borer, Chilo infuscatellus (Snellen) (Crambidae: Lepidoptera)

A total of eight field screened Erianthus arundinaceus genotypes against sugarcane shoot borer, Chilo infuscatellus was further screened under laboratory conditions along with a ruling variety Co 86032 for their confirmation of resistance and susceptibility. A significant reduction of C. infuscatellus larval and pupal survival; larval and female pupal weight; adult longevity; fecundity rate (eggs/female) and extended larval and pupal duration was recorded in the genotypes IJ 76 370, IJ 76 364 and IK 76 78, respectively. Biochemical parameters viz., total phenols, poly phenol oxidase and proteinase inhibitors and total proteins were highest in the genotypes IJ 76 370 and IJ 76 364 which indicates the presence of antibiosis mechanism of resistance against C. infuscatellus. The total soluble sugars were significantly lower in all genotypes. The genotypes IJ 76 364 and IK 76 84 fed – C. infuscatellus showed lowest larval total carbohydrate and protein content, respectively. The study identified two elite E. arundinaceus IJ 76 370 and IJ 76 364, that could be used as new genetic stocks for the possible utilization in the future resistant breeding programme against sugarcane shoot borer.

Molecular modeling and phenoloxidase inhibitory activity of arbutin and arbutin undecylenic acid ester

Excessive melanin formation has been linked to various skin disorders such as hyperpigmentation and skin cancer. Tyrosinase is the most prominent target for inhibitors of melanin production. In this study, we investigated whether arbutin and its prodrug, arbutin undecylenic acid ester, might inhibit phenoloxidase (PO), a tyrosinase-like enzyme. Molecular docking simulation results suggested that arbutin and arbutin undecylenic acid ester can bind to the substrate-binding pocket of PO. Arbutin undecylenic acid ester with an IC50 6.34 mM was effective to inhibit PO compared to arbutin (IC50 29.42 mM). In addition, arbutin undecylenic acid ester showed low cytotoxicity in Drosophila S2 cells and the compound inhibited the melanization reaction. Therefore, the results of this study have demonstrated that arbutin undecylenic acid ester as a potential inhibitor of PO. We successfully designed a new platform utilizing Drosophila melanogaster and Bombyx mori as animal models propounding fast, cheap, and high effectiveness in method to screen tyrosinase inhibitors.

Organic Carbon Stabilization Mechanisms in Mangrove Soils: A Review

Despite the recognized organic carbon (OC) sequestration potential of mangrove forests, the ongoing climate change and anthropogenic disturbances pose a great threat to these ecosystems. However, we currently lack the ability to mechanically understand and predict the consequences of such impacts, primarily because mechanisms underlying OC stabilization in these ecosystems remain elusive. Research into OC stabilization has focused on terrestrial soils and marine sediments for decades, overlooking the vegetated coastal ecosystems including mangroves. In terrestrial soils and marine sediments, it is widely accepted that OC stabilization is the integrated consequence of OM’s inherent recalcitrance, physical protection, and interactions with minerals and metals. However, related discussion is rarely done in mangrove soils, and recalcitrance of roots and high net ecosystem production (high primary production and low heterotrophic respiration) have been considered as a primary OC sequestration mechanism in mangrove peat and mineral soils, respectively. This review presents the available information on the mechanisms underlying OC stabilization in mangrove soils and highlights research questions that warrant further investigation. Primary OC stabilization mechanisms differ between mangrove peat and mineral soils. In mangrove mineral soils, physico-chemical stabilization processes are important, yet grossly understudied OC stabilization mechanisms. In mangrove peat, recalcitrance of mangrove roots and the inhibition of phenoloxidase under the anoxic condition may be the primary OC stabilization mechanisms. Salinity-induced OC immobilization likely plays a role in both type of soils. Finally, this review argues that belowground production and allochthonous inputs in mangrove forests are likely underestimated. More studies are needed to constrain C budgets to explain the enigma that mangrove OC keeps accumulating despite much higher decomposition (especially by large lateral exports) than previously considered.

Biochemical characterization of a hemolymph phenoloxidase and its endogenous inhibitor in the larvae of an invasive moth, Cydalima perspectalis Walker (Lepidoptera: Crambidae)

• A phenoloxidase was purified from the hemolymph of C. perspectalis with the molecular weight of ~ 78.7. • The optimal values of 7 and 30–35 °C were evaluated for pH and temperature dependency of the purified PO. • Syntehtic and endogenous inhibitors significantly affected PO activity both in vitro and in vivo. Phenoloxidase system is a crucial component of insect innate immunity which contribute to oxidize phenols to quinones and to generate reactive oxygen and nitrogen intermediates. In the current study, a phenoloxidase (PO) was extracted by hemocyte lysate preparation and purified through ammonium sulfate precipitation, Sepharyl G-100, and DEAE-Cellulose fast flow columns. At the end of the purification process, an enzyme was purified with a specific activity of 0.462 U/mg protein, recovery of 40.47%, purification fold of 14.43 and molecular weight of ~78.7 kDa. The optimal activity was recorded at pH 7 while the optimal temperature was recorded at 30–35 °C, 35 °C and 25–35 °C, using L-dihydroxyphenylalanine, hydroquinone, and pyrocatechol, respectively. The highest V max of PO was obtained using L-dopa while the lowest K m value was gained using hydroquinone. Among used synthetic inhibitors of ethylenediaminetetraacetic acid (EDTA), diethyldithiocarbamate (DTC), N, N,N0,N0-tetraacetic acid (EGTA) and triethylenetetramine hexaacetic acid (TTHA), EDTA and DTC inhibited more than 60% of the enzyme activity. Moreover, an endogenous phenoloxidase inhibitor (POI) was purified by twice processing of Sepharyl G-100 chromatography with the molecular weight of ~52 kDa. The IC 50 of POI was found 31.3 mg against the purified PO of C. perspectalis and led to a higher value of K m . Finally, larval injection by DTC and POI demonstrated significant inhibition of PO over the time of exposure. A comprehensive understanding of insect’s POs may better clarify the ways of their survival within infected areas and to potentially target them by specific and selective compounds.

Mild inactivation and inhibition of phenoloxidase in silkworm serum for xeno-free culture of insect cells

Mild inactivation and inhibition of phenoloxidase in silkworm serum for xeno-free culture of insect cells

Inhibition of Spodoptera frugiperda phenoloxidase activity by the products of the Xenorhabdus rhabduscin gene cluster.

We evaluated the impact of bacterial rhabduscin synthesis on bacterial virulence and phenoloxidase inhibition in a Spodoptera model. We first showed that the rhabduscin cluster of the entomopathogenic bacterium Xenorhabdus nematophila was not necessary for virulence in the larvae of Spodoptera littoralis and Spodoptera frugiperda. Bacteria with mutations affecting the rhabduscin synthesis cluster (ΔisnAB and ΔGT mutants) were as virulent as the wild-type strain. We then developed an assay for measuring phenoloxidase activity in S. frugiperda and assessed the ability of bacterial culture supernatants to inhibit the insect phenoloxidase. Our findings confirm that the X. nematophila rhabduscin cluster is required for the inhibition of S. frugiperda phenoloxidase activity. The X. nematophila ΔisnAB mutant was unable to inhibit phenoloxidase, whereas ΔGT mutants displayed intermediate levels of phenoloxidase inhibition relative to the wild-type strain. The culture supernatants of Escherichia coli and of two entomopathogenic bacteria, Serratia entomophila and Xenorhabdus poinarii, were unable to inhibit S. frugiperda phenoloxidase activity. Heterologous expression of the X. nematophila rhabduscin cluster in these three strains was sufficient to restore inhibition. Interestingly, we observed pseudogenization of the X. poinarii rhabduscin gene cluster via the insertion of a 120 bp element into the isnA promoter. The inhibition of phenoloxidase activity by X. poinarii culture supernatants was restored by expression of the X. poinarii rhabduscin cluster under the control of an inducible Ptet promoter, consistent with recent pseudogenization. This study paves the way for advances in our understanding of the virulence of several entomopathogenic bacteria in non-model insects, such as the new invasive S. frugiperda species in Africa.

Effect of endogenous phenoloxidase on protein solubility and digestibility after processing of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens

Upon extracting soluble proteins from insects as potential food ingredient, endogenous enzymes, such as phenoloxidases, are expected to negatively affect protein properties. The effect of phenoloxidases on solubility and digestibility of proteins was investigated for larvae of Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens. Phenoloxidase inhibition was done using blanching (50 s, 90 °C) before extraction or extracting in presence of sulfite.Similar soluble protein yields and compositions were found without and with sulfite addition, whereas blanching decreased soluble protein yield. Upon in-vitro hydrolysis by pepsin and trypsin, soluble proteins from H. illucens were more digestible than those of T. molitor and A. diaperinus. Phenoloxidase activity during grinding negatively affected in-vitro pepsin hydrolysis.Besides phenoloxidase activity, also endogenous proteases were shown to remain active at pH 8 in extracts containing sulfite and after blanching of larvae. This stresses that protease activity needs to be carefully controlled in the design of insect based ingredients.

Phenoloxidase, an effective bioactivity target for botanical insecticide screening from green walnut husks

Phenoloxidase, a critical enzyme in insects, may serve as a promising target in botanical insecticide development. In an effort to identify active ingredients with insecticidal properties in green walnut husks, juglone and plumbagin were isolated from the chloroform extract using phenoloxidase as bioactive target with the IC50 of 0.247 g/L and 0.256 g/L, respectively. After an artificial diet feeding of the juglone or plumbagin, more than 50% corrected mortality in stomach toxicity form was observed in Pieris rapae Linne larvae and Helicoverpa armigera Hübner larvae at the concentration ≥0.01 g/L, the LC50 of juglone and plumbagin for two kinds of insects were determined as 0.012, 0.011 and 0.022, 0.030 g/L, respectively. This research indicated the significance of PO as bioactive target in pesticides identification and also shed light on the development of phenoloxidase inhibitor as promising botanical insecticides in the future.

The activity of phenoloxidase in haemolymph plasma is not a predictor of Lymantria dispar resistance to its baculovirus.

Host innate immunity is one of the factors that determines the resistance of insects to their entomopathogens. In the research reported here we studied whether or not phenoloxidase (PO), a key enzyme in the melanogenesis component of humoral immunity of insects, plays a role in the protection of Lymantria dispar larvae from infection by L. dispar multiple nucleopolyhedrovirus. We studied two types of viral infection: overt and covert. The following lines of investigation were tested: i) the intravital individual estimation of baseline PO activity in haemolymph plasma followed by virus challenging; ii) the specific inhibition of PO activity in vivo by peroral treatment of infected larvae with phenylthiourea (PTU), a competitive inhibitor of PO; iii) the evaluation of PO activity in the haemolymph plasma after larval starvation. Starvation is a stress that activates the covert infection to an overt form. All of these experiments did not show a relationship between PO activity in haemolymph plasma of L. dispar larvae and larval susceptibility to baculovirus. Moreover, starvation-induced activation of covert viral infection to an overt form occurred in 70 percent of virus-carrying larvae against the background of a dramatic increase of PO activity in haemolymph plasma in the insects studied. Our conclusion is that in L. dispar larvae PO activity is not a predictor of host resistance to baculovirus.

Anti-Biofilm Effect of Biodegradable Coatings Based on Hemibastadin Derivative in Marine Environment.

Dibromohemibastadin-1 (DBHB) is an already known potent inhibitor of blue mussel phenoloxidase (which is a key enzyme involved in bioadhesion). Within this study, the potentiality of DBHB against microfouling has been investigated. The activity of DBHB was evaluated on key strains of bacteria and microalgae involved in marine biofilm formation and bioassays assessing impact on growth, adhesion and biofilm formation were used. To assess the efficiency of DBHB when included in a matrix, DBHB varnish was prepared and the anti-microfouling activity of coatings was assessed. Both in vitro and in situ immersions were carried out. Confocal Laser Scanning Microscopy (CLSM) was principally used to determine the biovolume and average thickness of biofilms developed on the coatings. Results showed an evident efficiency of DBHB as compound and varnish to reduce the biofilm development. The mode of action seems to be based principally on a perturbation of biofilm formation rather than on a biocidal activity in the tested conditions.

Purification and characterisation of a pronase-inducible lectin isolated from human serum

A new lectin was purified to electrophoretic homogeneity from pronase treated human serum by a single-step of affinity chromatography on concanavalin A-Sepharose 4B. The isolated lectin agglutinated five types of vertebrate RBC, with highest titer against hen RBC. This activity was independent of divalent cations, insensitive to EDTA and specific to mannosamine, glucosamine as well as galactosamine. Purified lectin gave a single symmetrical peak in its native form with a molecular mass estimate of 6kDa in FPLC analysis and 6.5kDa by MALDI-TOF MS. SDS-PAGE analysis of the lectin revealed that it is a homo-oligomer of a 3kDa subunit protein. Isolated lectin did possess both, hemagglutinating and phenoloxidase activities, but did not exhibit any antibacterial or antifungal activities. In addition, this lectin could oxidize all nine different phenolic substrates tested, with hydroquinone proving to be the best among them. Phenoloxidase inhibitors namely, phenylthiourea and tropolone inhibited this oxidation activity.

Postharvest control of litchi (Litchi chinensis Sonn.) pericarp browning by cold storage at high relative humidity after enzyme-inhibiting treatments

Enzyme inhibitors were studied as adjuvant treatments in the control of litchi pericarp browning by protection against desiccation during reefer transport. Various organic acids (acetic, malic, citric, and oxalic acid) and inorganic salts (NaCl, CaCl2) were investigated for in vitro inhibitory effects on the peroxidases (POD) and phenol oxidases (including laccase) extracted from litchi pericarp. Promising inhibitors were tested on ‘Hong Huey’ litchi fruit for their capability to prevent pericarp browning during cold storage (21 d, 5°C, 90% relative humidity) with and without foil wrapping. An enzyme assay had been optimized for measuring the in vitro activities of phenol oxidases toward (−)-epicatechin, being the natural phenolic key substrate in litchi. Phenol oxidase activities were chiefly pH-dependent and completely inactivated at pH≤3.5 by the organic acids used for buffering, whereby chelating agents performed best, especially oxalic acid. POD activity was stable over wider pH and ionic strength ranges, with inhibition being maximal (84%) in 0.25M oxalic acid buffer (pH 3.5). CaCl2 (0.25molL−1) decreased POD activity by 68%, while rising doses increased the initial lag phase up to 2.5 min. In contrast to these in vitro enzyme-inhibiting effects, postharvest fruit treatments with these phenol oxidase and peroxidase inhibitors did not improve color retention during cold fruit storage, but proved ineffective or even favored pericarp browning compared to the control fruit. Pericarp color retention was maximal (96–97%) throughout cold storage of fruit in gas-permeable but moisture-retaining foil bags for at least two weeks, whether the fruit had been dipped into cold water (control) or into citrate (25mmolL−1) solution. Consequently, pericarp color retention only required an intact pericarp at harvest and postharvest protection against desiccation. Preventing water loss through preservation of cell compartmentation thus proved to be crucial and sufficient for the control of enzymatic browning under reefer conditions.

From Insect to Man: Photorhabdus Sheds Light on the Emergence of Human Pathogenicity.

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called “nutritional virulence” strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

Dual effect of nitric oxide on phenoloxidase-mediated melanization

The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO2 at the active site of PO followed by oxidation of DOPA by NO2. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect’s haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.

SAR of Sponge-Inspired Hemibastadin Congeners Inhibiting Blue Mussel PhenolOxidase.

Hemibastadin derivatives, including the synthetically-derived 5,5′-dibromohemibastadin-1 (DBHB), are potent inhibitors of blue mussel phenoloxidase (PO), which is a key enzyme involved in the firm attachment of this invertebrate to substrates and, thus, a promising molecular target for anti-fouling research. For a systematic investigation of the enzyme inhibitory activity of hemibastadin derivatives, we have synthesized nine new congeners, which feature structural variations of the DBHB core structure. These structural modifications include, e.g., different halogen substituents present at the aromatic rings, different amine moieties linked to the (E)-2-(hydroxyimino)-3-(4-hydroxyphenyl)propionic acid, the presence of free vs. substituted aromatic hydroxyl groups and a free vs. methylated oxime group. All compounds were tested for their inhibitory activity towards the target enzyme in vitro, and IC50 values were calculated. Derivatives, which structurally closely resemble sponge-derived hemibastadins, revealed superior enzyme inhibitory properties vs. congeners featuring structural moieties that are absent in the respective natural products. This study suggests that natural selection has yielded structurally-optimized antifouling compounds.

Benzaldehyde as an insecticidal, antimicrobial, and antioxidant compound produced by Photorhabdus temperata M1021.

The Photorhabdus temperata M1021 secretes toxic compounds that kill their insect hosts by arresting immune responses. Present study was aimed to purify the insecticidal and antimicrobial compound(s) from the culture extract of P. temperata M1021 through bioassay guided fractionation. An ethyl acetate (EtOAc) extract of the P. temperata M1021 exhibited 100% mortality in Galleria mellonella larvae within 72 h. In addition, EtOAc extract and bioactive compound 1 purified form the extract through to column chromatography, showed phenol oxidase inhibition up to 60% and 80% respectively. The analysis of (1)H and (13)C NMR spectra revealed the identity of pure compound as "benzaldehyde". The benzaldehyde showed insecticidal activity against G. mellonella in a dose-dependent manner and 100% insect mortality was observed at 108 h after injection of 8 mM benzaldehyde. In a PO inhibition assay, 4, 6, and 8 mM concentrations of benzaldehyde were found to inhibit PO activity about 15%, 42%, and 80% respectively. In addition, nodule formation was significantly (P < 0.05) inhibited by 4, 6, and 8 mM of benzaldehyde as compare to control. Moreover, benzaldehyde was found to have great antioxidant activity and maximum antioxidant activity was 52.9% at 8 mM benzaldehyde as compare to control. Antimicrobial activity was assessed by MIC values ranged from 6 mM 10 mM for bacterial strains and 8 mM to 10 mM for fungal strains. The results suggest that benzaldehyde could be applicable for developing novel insecticide for agriculture use.

An insecticidal compound produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition.

A bioassay-guided column chromatographic strategy was adopted in the present study to fractionate the culture extract of Photorhabdus temperata M1021 to identify potential insecticidal and antimicrobial compounds. An ethyl acetate (EtOAc) culture extract of P. temperata was assayed against Galleria mellonella larvae through intra-hemocoel injection and exhibited 100% insect mortality within 60 h. The EtOAc fraction and an isolated compound exhibited phenoloxidase (PO) inhibition of up to 60% and 63%, respectively. The compound was identified as 1,2-benzenedicarboxylic acid (phthalic acid, PA) by gas chromatography-mass spectrometry and nuclear magnetic resonance. PA exhibited insecticidal activity against G. mellonella in a dose-dependent manner, and 100% insect mortality was observed at 108 h after injection of 1 M PA. In a PO inhibition assay, 0.5 and 1 M concentrations of PA were found to inhibit PO activity by 74% and 82%, respectively; and in a melanotic nodule formation assay, nodule formation was significantly inhibited (27 and 10 nodules) by PA (0.5 and 1 M, respectively). PA was furthermore found to have substantial antioxidant activity and maximum antioxidant activity was 64.7% for 0.5 M PA as compare to control. Antibacterial activity was assessed by The MIC values ranged from 0.1 M to 0.5 M of PA. This study reports a multifunctional PA, a potential insecticidal agent, could a factor of insect mortality along with other toxins produced by P. temperata M1021.

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

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 1.17 U/mg protein, recovery of 20.48% and purification fold of 16.71. The purified PO showed the highest activity at pH 4–5 and temperatures of 35–40 °C. Na+, K+, Mn+, Zn2+ and Mg2+ decreased activity of the purified PO but Ca2+ and Cu2+ 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 Vmax when L-DOPA was used as substrate but no significant differences were observed in case of Km for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC50 of 96.41 and 38.59 µg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC50 concentrations of the two IGRs revealed the increase of Km 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.