Phenoloxidase Inhibitors Research Articles

EPR Detection of Reactive Oxygen Species in Hemolymph of Galleria mellonella and Dendrolimus superans sibiricus (Lepidoptera) Larvae

The formation of reactive oxygen species (ROS) in hemolymph and hemocytes of Galleria mellonella and Dendrolimus superans sibiricus larvae was studied by ESR spectroscopy using spin-trap 1-hydroxy-3-carboxy-pyrrolidine (CP-H). The background level of ROS formation was detected in the intact hemolymph. The addition of dihydroxyphenylalanine (DOPA) into free cells of the hemolymph increased CP-H oxidation about two times for G. mellonella and about four times for D. superans sibiricus. This increase was completely inhibited by a specific inhibitor of phenoloxidase, phenylthiourea. The presence of exogenous superoxide dismutase (SOD) did not change CP-H oxidation in the hemolymph. The data obtained in hemocytes showed only a DOPA-induced increase in CP-H oxidation. Phagocytosis activators did not affect ROS formation in hemocytes of both insect species. SOD decreased DOPA-induced CP-H oxidation 20–30% in suspension of hemocytes of D. superans sibiricus only. Our results are in agreement with the contribution of superoxide radical and DOPA-derived quinones/semiquinones in the immune response of insects.

A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins.

Phenoloxidase inhibitor (POI), found in the hemolymph of housefly pupae, is a novel dopa-containing and cystine-rich peptide that competitively inhibits phenoloxidase with a Ki in the nanomolar range. [Tyr32]POI is a potential precursor molecule also found in the hemolymph that may be posttranslationally oxidized to the dopa-containing peptide after creation of a rigid structure. By employing both a solid-phase peptide synthesis system based on a 9-fluorenylmethoxycarbonyl strategy and a specific air oxidation technique to ensure correct folding, we have been able to synthesize [Tyr32]POI. The synthetic [Tyr32]POI was confirmed to be identical to the native [Tyr32]POI by coelution high-performance liquid chromatography analysis and by enzymatic analysis using the phenoloxidase inhibition assay. To determine the disulfide pairings within the peptides, a series of enzyme hydrolyses and partial reduction/alkylation steps were performed. Three cystine pairs (Cys11-Cys25, Cys18-Cys29, and Cys24-Cys36) were determined by identification of the resulting peptides. The disulfide pairings of the two adjacent Cys residues (Cys11-Cys25 and Cys24-Cys36) were unambiguously assigned by comparing the derived fragments with the two possible isomers synthesized through a novel disulfide-linking technique. The arrangement of the disulfide bridges in POI was found to be topologically identical to those found for several peptides within the inhibitor cystine knot structural family. Although these peptides share a low primary sequence homology and display a diversity of biological functions, they nonetheless share similarities in their cystine motifs and tertiary structure. The tertiary structure model of POI, which was derived through molecular dynamics and energy minimization studies using restraints with determined disulfide connectivities, suggests that POI is a new class member of the inhibitor cystine-knot structural family.

Noxious Components of Venom from the Pupa-Specific ParasitoidPimpla hypochondriaca

Venom from the endoparasitoid waspPimpla hypochondriacainduced a lethal paralysis when injected into the hemocoel of larval, pupal, and adult stages of the tomato mothLacanobia oleraceaand was also toxic to the adult stages of the houseflyMusca domesticaand the cockroachBlatella germanica.Heating the venom to 58°C did not reduce its potency againstM. domestica.Phenoloxidase activity was detected in diluted venom using dihydroxyphenylalanine as the substrate and occurred independently of activators. Enzyme activity was strongly inhibited by 2.0 μM phenylthiocarbamide (PTC), a known inhibitor of phenoloxidase, and was not detectable in venom that had been heated to 58°C for 10 min. Venom treated to remove phenoloxidase was shown to inhibit phenoloxidase activity in untreated venom, with a potency likely to be sufficient to prevent enzyme activity in the parasitoid's venom sac. The venom was found to be cytotoxic to an insect cell line (SF21) fromSpodoptera frugiperda,and this activity was not reduced when the cells were cultured in the presence of 20 μM PTC. Venom constituents were separated according to size using high-resolution gel filtration. Three venom components: phenoloxidase, a factor which caused paralysis ofM. domestica,and a factor which was cytotoxic to SF 21 cells were detected and found to have maximum activity in fractions 19, 40–42, and 50–51, respectively. Phenoloxidase and the factor causing paralysis ofM. domesticahad estimated apparent molecular masses of 390 and 27 kDa, respectively, while the cytotoxic factor had an apparent molecular mass of less than 13 kDa. Assay for inhibitors of venom phenoloxidase identified two major inhibitory components having highest activity in fractions 48 and 60, respectively. The larger of these two factors had an estimated apparent molecular mass of 12 kDa, but the other factor was considerably smaller than the lowest molecular mass standard (13.7 kDa) used to calibrate the column.

Properties of phenoloxidases generated from prophenoloxidase with 2-propanol and the natural activator in Drosophila melanogaster.

Prophenoloxidases A1 and A3 in Drosophila melanogaster were activated with 2-propanol and a partially purified natural activator. For prophenoloxidase activation, the optimum temperature was 30 degrees C and the optimum pH was 8. Both mono- and diphenoloxidase activities were found in A1 and A3 activated with 2-propanol, whereas only diphenoloxidase activity was detected in A3 activated with a natural activator. The kinetic properties, Km and Vmax, were not similar in those phenoloxidases activated with different activating agents. The rate of inhibition of phenoloxidase by diethyldithiocarbamate and phenylthiocarbamate depended on the concentration of 2-propanol. Both compounds exhibited a noncompetitive pattern of inhibition.

Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.

Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica.

A mechanism for eggshell production in Schistosoma mansoni has been proposed (Wells & Cordingley, 1991), and suggests that the release of eggshell protein globules from the vitelline cells occurs under alkaline conditions within the ootype followed by their subsequent fusion to form the eggshell. Fusion and tanning of these components produces eggshell which autofluoresces. The present study was carried out to determine whether a similar process operates in Fasciola hepatica. A number of drug treatments were used to disrupt key steps in the maturation of vitelline cells. Treatment with the calcium ionophore lasalocid (1 x 10(-5) M) led to the premature release of eggshell globules from the vitelline cells but not their fusion. Incubation in monensin (1 x 10(-6)M), a sodium ionophore and ammonium chloride (NH4Cl) (5 x 10(-2) M), a weak base, resulted in the premature fusion of eggshell protein globules within the vitelline cells and premature tanning of the eggshell protein material. The copper-containing enzyme, phenol oxidase, is thought to be involved in the tanning process during the production of eggs. Diethyldithiocarbamate, (DDC, 1 x 10(-3) M) is a phenol oxidase inhibitor and treatment with this compound, in combination treatments with monensin and NH4Cl, prevented fusion of the vitelline cell globules and tanning of the shell protein material. The results of the study suggest that the mechanism for eggshell formation in F. hepatica is similar to that proposed for S. mansoni and may be common to other trematodes as well.

Phenoloxidase Activity in the Reproductive System of Biomphalaria glabrata: Role in Egg Production and Effect of Schistosome Infection

Infection by larval trematodes often causes a cessation of egg production in its molluscan intermediate host and is referred to as parasitic castration. Because phenoloxidase (PO) has been shown to be involved in egg formation in other invertebrate species, we investigated the role of PO in normal egg production in the snail, Biomphalaria glabrata, and the effects of Schistosoma mansoni infection on the PO pathway in this snail. Our data showed that PO activity in the albumen gland (AG) is initially expressed when snails reach a size of approximately 8 mm in shell diameter and continues to increase as snails grow, indicating a developmental link between snail size and AG PO expression. Egglaying was also shown to be coincidental with the onset of PO expression in the AG, thereby supporting a direct association between PO activity and egg production. In addition, exposure of snails to diethyldithiocarbamate (DDC), a PO inhibitor, affected normal in vivo egg production, as evidenced by a significant decrease in the numbers of eggs laid in DDC-treated groups compared to nontreated groups. Normal resumption of egg-laying activity in treated snails following withdrawal of the drug indicated that inhibition was reversible. Taken together, the results of our developmental and DDC-exposure studies provide strong support for a crucial role of PO in normal egg production in this animal. Finally, AG PO activities of infected and uninfected control snails were measured over the course of S. mansoni infection. Our results showed that both total and specific enzyme activities in the AG of infected snails were significantly decreased at 28 and 33 days postinfection (PI) when compared to those of control snails. Results of subsequent experiments assessing the effects of larval infection on L-tyrosine (PO substrate) levels in AG and ovotestis revealed a significant increase in the levels of this compound in both organs over the course of infection. It is concluded that AG PO activity is functionally linked to egg formation in normal snails and that a strong association exists between parasite-mediated decrease in AG PO activity and parasitic castration. However, from the data presented, a direct causal relationship linking infection, decreased PO, and castration has yet to be established.

The Effect of Disulfiram in Egg Shell Formation in Adult Trichuris muris

The effect of disulfiram on egg shell morphology in the parasitic nematode Trichuris muris was studied in vitro and in vivo. Daily disulfiram treatment of mice infected with T. muris beginning 25 days after infection and continuing for 26 days resulted in the production of malformed eggs by adult female worms in all treated groups. In addition, significantly fewer adult worms were found at necropsy in mice treated with 5.0 or 7.5 mg/kg/day of disulfiram as compared with mice treated with 2.5 mg/kg/day or control mice. Adult worms collected from infected, untreated mice and placed in aerobic culture for 5 days in media containing 4 or 8 micrograms/ml of disulfiram released malformed eggs into the culture medium after 30 hr in culture. Oral inoculation of naive mice with malformed eggs did not result in T. muris infections in the mice. The results of these studies suggest that inhibition of phenol oxidase results in disruption of normal egg production by T. muris females and that the enzyme might be a useful target in the development of control strategies aimed at nematode parasites that rely on phenol oxidase for egg shell hardening.

Conversion of phenoloxidase and peroxidase indicators in individual haemocytes of Mytilus edulis specimens and isolation of phenoloxidase from haemocyte extract

Haemocytes oxidized 3-amino-9-ethylcarbazole and other peroxidase indicators such as 3,3′-diaminobenzidine·4HCl, 3,3′,5,5′ tetramethylbenzidine·2HCl and 4-chloro-1-naphthol without addition of H2O2 indicating that the reaction was possibly not caused by a peroxidase. As these chromogens were also converted by a mushroom phenoloxidase in the absence of H2O2, cell smears were incubated with known substrates of phenoloxidases. One of these, l-dopa, caused strong melanin formation in several haemocytes and the reaction could be blocked by a variety of inhibitors including KCN, NaF, 1-phenyl-2-thiourea, cysteine, glutathione, ascorbic acid and HgCl2. The enzymatic activity was isolated using a concanavalin A column and separated into two fractions with an ion-exchange cartridge. The molecular weights of the glycoproteins were estimated to be 381±13.7 kDa and 316±11.1 kDa. After isoelectric focusing of a haemocyte extract and the two ion-exchange peaks, seven enzyme bands were detected with isoelectric points between pH 5.0 and 5.5. The isolated enzyme fractions both converted 3,3′,5,5′-tetramethylbenzidine·2HCl best at pH 5–6 and l-dopa at pH 7.0 without addition of H2O2. Heat-treated cells lost their enzymatic activity; however, a group of haemocytes still bound preoxidized 3-amino-9-ethylcarbazole (= AECox). Also, some of the phenoloxidase inhibitors mentioned above blocked this non-enzymatic staining reaction. About 30–57% of haemocytes from individual mussels were AECox-positive, whereas Mytilus specimens without phenoloxidase-containing cells often occurred. Haemocytes containing this enzyme exhibited a high mobility and a large percentage of them belonged to a cytotoxic cell population.

Latent larval cuticular phenoloxidase in the coconut pest,Oryctes rhinoceros

Cuticular phenoloxidase (Tyrosinase) in the larval stage of the coconut pest Oryctes rhinoceros has been extracted in the stable proform using cane sugar saline/borate buffer. The extracted prophenol oxidase can be activated by the addition of proteolytic enzymes such as trypsin, chymotrypsin, thermolysin, and subtilisin. Detergents such as SDS and Tween-80 also activated the enzyme. Electrophoretical analysis revealed dissociation of the enzyme into two molecular forms after its activation by proteolytic enzymes. The functional significance of the enzyme is suggested to involve the generation of quinone compounds in the wound healing process: most phenoloxidase inhibitors prevented melanization when applied topically to surgical wounds. © 1996 Wiley-Liss, Inc.

Wounding increases intracellular encapsulation (melanization) of developing Brugia malayi (Nematoda: Filarioidea) larvae in thoracic muscles of Anopheles quadrimaculatus

Wounding of females of the refractory strain of Brugia malayi-infected Anopheles quadrimaculatus by an injection of saline solution resulted in a significant increase in encapsulation and melanization of intracellularly located first stage (L 1) larvae of B. malayi in their thoracic muscles 24 and 48 hr after the injection, when compared to uninjected infected females. This increased encapsulation and melanization response was inhibited by injecting saline solution containing prophenoloxidase and phenoloxidase inhibitors and competitors (benzamidine HCl, p-nitrophenyl- p′guanidinobenzoate HCl, soybean trypsin inhibitor, diethyldithiocarbamic acid-sodium salt, 1-phenyl-2-thiourea, and reduced glutathione) into B. malayi-infected An. quadrimaculatus females. These results demonstrate for the first time that wounding of the cuticle by injecting saline solution into the hemocoel of the infected females activated the prophenoloxidase cascade that increased encapsulation and melanization of intracellularly located L 1 of B. malayi in infected thoracic muscles of An. quadrimaculatus.

Primary structure of a potent endogenous dopa-containing inhibitor of phenol oxidase from Musca domestica.

The complete amino acid sequence of a low molecular weight peptide from the hemolymph of the housefly Musca domestica L., which had been determined to competitively inhibit phenol oxidase (PO; monophenol, dihydroxy-phenylalanine:oxygen oxidoreductase; EC 1.14.18.1) in the nM range, was unambiguously established by employing both automatic Edman degradation and mass spectrometry. The physiologically active peptide, which was designated phenol oxidase inhibitor (POI), has an observed molecular weight of 4213.1 +/- 0.2 by electrospray ionization mass spectrometry. The relatively short and structurally dense peptide contained 38 amino acid residues rich in cysteine and lysine. Comparison of the observed and calculated molecular mass indicates that apparently all six cysteine residues form disulfide bridges. Interestingly, sequence analyses of both the intact and protease-digested S-pyridylethylated POI showed that one of the two tyrosine residues (Tyr-32) is hydroxylated to a 3,4-dihydroxyphenylalanine (dopa) residue. This agreed with the increase of 16 mass units observed in mass spectrometric measurements. This was further verified by submission of free L-dopa to the sequencer, which gave a retention time consistent with the atypical peak observed at the Edman cycle of the peptide containing dopa. This study demonstrates the existence of a biologically active, dopa-containing peptide among the insects. Since the POI activity was most prominent in aged pupae, especially pharate adults, the POI may play an important role in smoothing the way of adult emergence through hindering excessive melanization, as well as hardening, of cuticular proteins under the epicuticle.

Comparative Study of Substrates and Inhibitors of Azospirillum lipoferum and Pyricularia oryzae Laccases

Azospirillum lipoferum and Pyricularia oryzae laccases were compared, using several substrates and inhibitors. Sixteen phenolic or nonphenolic compounds were found to be substrates of both fungal and bacterial laccases. In the presence of different phenol oxidase inhibitors, P. oryzae and A. lipoferum laccase activities had similar properties.

Localization of Phenol Oxidase in Female Trichuris suis

A phenol oxidase (E.C. 1.10.3.1) preparation from adult female Trichuris suis was assayed by both polarographic and spectrophotometric techniques. The T. suis enzyme oxidized most diphenols including 4-methylcatechol (4MC) and dihydroxyphenylalanine but did not oxidize tyrosine. The pH and temperature optima were 6.8 and 36 C, respectively. The Km measured using 4MC as a substrate ranged from 0.12 to 0.4 mM. The highest phenol oxidase activity was isolated in fractions from the adult females that were enriched in eggs relative to the activity in somatic tissue from females and all male tissues that were assayed. Phenol oxidase activity was localized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis substrate gels into 2 bands with M(r)'s of 44,000 and 53,000. An antibody to the 44,000 band recognized 2 bands of 40,000 and 45,000 M(r) on western blot analysis of the enzyme preparation. Immunocytochemical localization of anti-phenol oxidase antibody in serial cross sections of adult female worms indicates that the enzyme is found exclusively in the anterior part of the parasite in the proximal part of the uterus that is posterior to the junction with the stichosome. Eggs located in more distal parts of the reproductive system did not react with the antibody. The results indicate that a phenol oxidase is located in the fertilized eggs of adult female T. suis. It is likely that phenol oxidase contributes significantly to the chemical hardening process in the eggs when they pass out into the external environment. Inhibition of phenol oxidase may reduce the survivability of the eggs and thus minimize contamination of livestock facilities.

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.

Role of Tyrosine Hydroxylase in the Blood of the Ghost Crab DcΥPode PlatΥTarsis H. Milne Edwards, 1852 (Decapoda, Brachyura)

The blood of Ocypode platytarsis contains a single fraction of tyrosine hydroxylase (E.C.1.14.16.2), which is capable of converting the monophenol into O-diphenol and 3 fractions of phenoloxidase (E.C.1.14.18.1), which convert O-diphenol into quinone. The phenoloxidases do not show any monophenoloxidase activity. The phenoloxidase fractions exist as proenzyme and can be activated by sodium dodecyl sulphate. Tyrosine hydroxylase may be involved in the synthesis of O-diphenol. The stored O-diphenol may be oxidised into melanin during immunological response, after the activation of prophenoloxidase. Thin layer chromatographic analysis reveals that there is an accumulation of dopa methyl ester in the reaction medium containing the enzyme sample, tyrosine methyl ester and phenylthiourea (PTU), a specific inhibitor of phenoloxidase. In the absence of PTU, the reaction medium becomes brown and there is no accumulation of O-diphenol.

Identification and isolation of endogenous insect phenoloxidase inhibitors

Inhibitors of phenoloxidase were identified in pupae of the housefly, Musca domestica L. The phenoloxidase inhibitors were purified from final instar pupae of the housefly by a combination of ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and reverse-phase high performance liquid chromatography. The potent phenoloxidase inhibitors were heat-stable low molecular weight peptides with an inhibition constant of nM range. To the best of our knowledge, this is the first time that endogenous phenoloxidase inhibitors have been identified among the insects, and probably also among the invertebrates. It is likely that the inhibitors play a central role in regulating the action of active phenoloxidases and will also serve as important tools for understanding the structures and functions of phenoloxidases, as well as their role in insect metamorphosis.

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.

DOPA oxidation by Holothuria polii coelomocyte lysate

Phenoloxidase activity was demonstrated in coelomocyte lysate (CL) from Holothuria polii, but not in cell free coelomic fluid. Oxidation of DOPA to dopachrome was measured photometrically after activation of CL with trypsin or chymotrypsin. Chymotrypsin activation of CL was inhibited by serine protease inhibitors such as STI and PMSF. β-1,3-glucans (incubation of CL with zymosan or laminarin) also triggered DOPA oxidation activity of CL but the effect was not as strong as with chymotrypsin. Addition of the phenoloxidase inhibitor PTU completely suppressed DOPA oxidation in CL. The role of prophenoloxidase activation in immune reactions of invertebrates is discussed.

Evidence and cellular localization of an oxidative activity in the coelomic fluid of the earthworm Eisenia fetida andrei

An oxidative activity was evidenced in the coelomic fluid of the earthworm Eisenia fetida andrei, when l-DOPA was used as substrate. Oxidative activity, measured by optical density due to dopachrome formation, was found to be optimal at basic pH (9–10). Changes in the rate of substrate conversion observed after the addition of hydrogen peroxide or catalase suggest that oxidation of l-DOPA by E. fetida andrei coelomic fluid is mediated by both oxidasic and peroxidasic systems. Microscopy studies, using substrates for the determination of both phenoloxidase and peroxidase activities, demonstrated that the enzyme(s) is localized in chloragocyte vesicles called chloragosomes. The persistence of a remnant staining after incubation with phenoloxidase inhibitors as well as with peroxidase inhibitors strengthened the hypothesis of a synergistic activity of two oxidative systems in E. fetida andrei coelomic fluid.