Monophenol Oxidase Activity Research Articles

Functional analysis of walnut polyphenol oxidase gene (JrPPO1) in transgenic tobacco plants and PPO induction in response to walnut bacterial blight

AbstractWalnut (Juglans regia) is economically important for both its wood and nut nutritional value, but it is susceptible to diseases such as walnut bacterial blight, caused by Xanthomonas arboricola pv. juglandis (Xaj). Walnuts contain many phenolic compounds, providing a good model on which to study polyphenol oxidase (PPO). We inoculated the detached walnut fruits of cultivars Ford, Chandler, Franquette, Robert Livermore, and Payne with Xaj and measured the induction of PPO activity in infected sites and adjacent to infected sites. Compared to infected and uninfected sites, PPO activity was induced significantly in areas adjacent to infected sites in all cultivars except Ford. Ford and Franquette, presenting the lowest and highest PPO activity, showed the largest and smallest mean diameter spots in response to Xaj, respectively. Polyacrylamide gel electrophoresis confirmed monophenol oxidase activity of walnut PPO in the assessed tissues. Then, we revealed the antipathogenic potential of walnut PPO through Agrobacterium tumefaciens‐mediated walnut JrPPO1 gene transfer into tobacco (Nicotiana tabacum). Two transformed tobacco lines overexpressing the JrPPO1 gene were regenerated successfully and challenged with Pseudomonas syringae pv. tabaci. Transgenic lines showed significantly higher PPO activity and lower disease severity to the pathogen compared to the control. However, a significant difference in disease severity and PPO activity level was observed between the two transgenic lines. Our results demonstrate a potential defence‐related role of PPO in transgenic tobacco and its induction in areas adjacent to infection sites in walnut cultivars treated with Xaj.

Molecular and biochemical characterization of Paragonimus westermani tyrosinase.

Trematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5.0 to 8.0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

Electrophoretic Separation and Identification of Phenoloxidases in Hemolymph and Midgut of Adult Anopheles stephensi Mosquitoes

Anopheles mosquitoes frequently respond to invading malaria parasites with a rejection mechanism consisting of phenoloxidase-mediated melanization of ookinetes in the mosquito midgut epithelium. The relative roles of hemolymph vs. midgut phenoloxidase in this rejection mechanism is unclear. We have separated and identified phenoloxidase isozymes from midgut and hemolymph of Anopheles stephensi by native gel electrophoresis followed by zymography. The isozymes from the 2 sites had distinctively different electrophoretic characteristics. Hemolymph possessed 2 phenoloxidase-positive bands, both of which were bifunctional molecules that oxidized monophenol as well as o-diphenol substrates. Midgut extract possessed 3 bands that migrated more rapidly than those of the hemolymph. None of these midgut bands had detectable monophenoloxidase activity; they possessed, however, a broad spectrum of diphenoloxidase activity in their ability to oxidize both o- and p-diphenol substrates, as well as the laccase substrate syringaldazine. The 2 most rapidly migrating midgut PO bands could be distinguished from the more slowly migrating band through their insensitivity to inhibition by the chelating agent tropolone. A question to be resolved in the future relates to the relative roles of hemolymph vs. midgut phenoloxidase in mosquito defense against invasive parasites.

Phenoloxidase activity in the reproductive system and egg masses of the pulmonate gastropod, Biomphalaria glabrata

Phenoloxidase (PO) activity in the albumen gland (AG) and egg masses (EM) of Biomphalaria glabrata was assessed using high-performance liquid chromatography combined with electrochemical detection and colorimetric techniques. Both AG and EM extracts catalyzed the hydroxylation of l-tyrosine (monophenol oxidase activity, MPO) and oxidation of l-dopa (diphenol oxidase activity, DPO). However, no PO activity was found in the ovotestis. Both MPO and DPO activities in AG and EM were significantly inhibited by 1-phenyl-2-thiourea and inactivated by boiling. Approximately 35% of MPO and 44% of DPO activities were detected in the soluble fraction of homogenized EM, in contrast to that of homogenized AG, which contained about 5% and 12%, respectively, of MPO and DPO activities. N-acetyl-dopamine, a diphenolic compound, enhanced the hydroxylation of tyrosine by the PO. The presence of both MPO and DPO activities also was confirmed by the accelerated accumulation of dopachrome during incubation of EM extracts with l-tyrosine in the absence of ascorbate. Temperature and pH optima for this enzyme were 30°C and 7.5, respectively. The potential roles of PO in egg formation in B. glabrata are discussed.

Observations on the Germ Aleurone of Barley. Phenol Oxidase and Peroxidase Activity

Strips of tissue containing the germ aleurone layer were removed from dry, harvest-ripe grains of barley (Hordeum vulgare L.) and incubated in buffered solutions of phenolic compounds, with and without the addition of hydrogen peroxide. Peroxidase and o-diphenol oxidase activity were found in the material released into the incubation medium, and in the cytoplasm of the germ aleurone cells. Peroxidase activity was located in the cell walls and appeared to be high in the region where the germ aleurone covering the embryonic axis merges into that which adheres to the scutellum i.e. the region in which a row of germ aleurone cells becomes lignified following germination. Monophenol oxidase activity was not detected in the released enzymes or in the intact tissue. Although hydroquinone was oxidized in the cytoplasm of the germ aleurone tissue, unequivocal evidence of the presence of laccase was not obtained. The oxidation of endogenous phenolic substances by phenol oxidases and peroxidases is discussed in relation to anti-microbial defence mechanisms which appear to operate in the germ aleurone during germination.

Monophenol oxidase activity from the cuticle of Florida spiny lobster (Panulirus argus)

Endogenously activated phenoloridase (PO) from the cuticle of Florida spiny lobster was purified 37-fold. It had an isoelectric point of 4.76 and an apparent molecular weight of 81 200 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion-pair reversed-phase high-performance liquid chromatography demonstrated that PO can catalyze the hydroxylation of L-tyrosine to dihydroryphenylalanine (mono-PO activity). The optimum pH for hydrorylation was between 6.0 and 6.5, and the PO was most stable from pH 6 to 8. The optimum temperature for reaction was 40 o C, and PO was stable up to 40 o C. The apparent Michaelis-Menten constant (K m ) was 0.97 mM for PO hydrorylation of L-tyrosine

Characteristics of Phenol Oxidase of Schistosoma mansoni and Its Functional Implications in Eggshell Synthesis

It is proposed that the phenol oxidase enzyme of schistosomes and other trematodes has a crucial role in the cross-linking of precursor proteins in the formation of the eggshell. However, to date there is no direct evidence to show that the enzyme catalyzes the reactions necessary for the posttranslational modification of eggshell precursor proteins. In this report we demonstrate that an eggshell precursor protein acts as a substrate for a schistosome fraction that catalyzed the 2 steps in the oxidation of tyrosine. This action of the phenol oxidase-containing worm fraction resulted in the tyrosine-dependent insolubilization and aggregation of the protein, suggesting a role for the enzyme in the posttranslational modification and cross-linking of schistosome eggshell proteins. The enzyme-rich fraction from Schistosoma mansoni catalyzed both steps of the reactions necessary for the conversion of tyrosine residues on putative eggshell precursor protein (p48) to quinones. The parasite fraction also catalyzed the hydroxylation of free L-tyrosine to DOPA (monophenol oxidase activity) and the oxidation of L-DOPA to dopaquinone (diphenol oxidase activity). Both activities of the enzyme are avidly bound to membranous structures, are susceptible to agents known to inhibit a functionally related enzyme, tyrosinase, and may reside on the same protein.

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.

Relationship of hemolymph phenol oxidase and mosquito age in Aedes aegypti

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity in hemocytes and cell-free plasma perfused from 7-, 14-, 21-, and 28-day-old Aedes aegypti mosquitoes were compared. A progressive decrease of enzyme activity was detected as mosquito age increased, and this decrease was significant in both hemocytes and cell-free plasma when mosquitoes were 28 days old as compared with that found in 7-day-old mosquitoes. There was no significant difference in total hemolymph protein as mosquito age increased. Although this decreased MPO and DPO activity might be partially responsible for the reduced immune response against filarial worms previously reported for older mosquitoes, other factors undoubtedly play a significant role.

Phenol oxidase activity in hemolymph compartments of Aedes aegypti during melanotic encapsulation reactions against microfilariae

Monophenol oxidase (MPO) and diphenol oxidase (DPO) activity was assessed in hemocytes, cell-free plasma and complete hemolymph collected from Aedes aegypti Liverpool strain, intrathoracically inoculated with saline alone, immune activated by the inoculation of Dirofilaria immitis microfilariae (mff), and uninoculated. Enzyme activities between groups were compared using a radiometric hydroxylation assay (MPO) and a high pressure liquid chromatography with electrochemical detection assay (DPO). There were no significant differences in enzyme activity in hemocytes, cell-free plasma, and complete hemolymph between uninoculated and saline-inoculated controls. Both MPO and DPO activity of mosquito hemocytes and complete hemolymph from immune-activated mosquitoes were significantly increased at 12 and 24 h postinoculation as compared with the enzyme activity from saline-inoculated mosquitoes, but no significant increase in enzyme activity was detected in cell-free plasma from immuneactivated mosquitoes. Increases of MPO and DPO activity in hemocytes and hemolymph following immune activation were proportional, thereby suggesting that a single enzyme might react with both monophenols and o-diphenols within the hemolymph of A. aegypti. Results also suggest that augmented phenol oxidase activity associated with melanotic encapsulation reactions is associated primarily with hemocytes.

Hemolymph phenol oxidases in Drosophila melanogaster, Locusta migratoria, and Austropotamobius pallipes

The early enzyme-mediated reaction sequence in the biosynthesis of melanin from L-tyrosine involves an initial hydroxylation (monophenol oxidase activity, MPO) of the aromatic amino acid precursor to form L-dopa (3,4-dihydroxyphenylalanine), and the ensuing oxidation (diphenol oxidase activity, DPO) of the resultant diphenol to form dopaquinone. By means of high pressure liquid chromatography with electrochemical detection (HPLC-ED) both phenol oxidase activities were observed in the blood (hemolymph) of two species of insect, third-stage larvae of Drosophila melanogaster and adult Locusta migratoria, and in an adult fresh-water crayfish, Austropotamobius pallipes. These results establish that in each species MPO and DPO can be detected readily without the use of exogenous activators.

Analysis of Monophenol Oxidase Activity Using High Pressure Liquid Chromatography with Electrochemical Detection

Abstract A very sensitive, specific and reliable quantitative assay was developed for measuring the rate of hydroxylation of tyrosine by mushroom tyrosinase using high pressure liquid chromatography with electrochemical detection (HPLC-ED). The assay employs N-acetyldopamine (NADA) as cofactor and ascorbate as a reducing agent. The product of the reaction, L-dopa (3,4-dihydroxyphenylalanine), was readily separated by HPLC-ED from the remaining interacting components. The reaction was linear with time and proportional to the amount of enzyme present. The amount of ascorbate gradually decreased during the hydroxylation of tyrosine, but as long as ascorbate was present in the reaction mixture the levels of L-dopa and NADA were not altered. The data

Haemolymph monophenol oxidase activity in Armigeres subalbatus infected with Brugia pahangi

The activity of monophenol oxidase can be elicited in the haemolymph of Armigeres subalbatus by both blood and filariainfected blood feeding. Haemolymph collected from both blood-fed and filaria-infected mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of l-[3,5- 3H]tyrosine to dopa. Enzyme activity in filaria-infected mosquitoes was found to be significantly lower than that found in the blood-fed mosquitoes within 3 days post-ingestion, but still remained measurable 72 h post-ingestion. The decreased enzyme activity coincided in time with the development of capsules around the microfilariae. The consumption of monophenol oxidase by the melanization of migrating microfilariae in the haemocoel of filaria-infected mosquitoes and the effects of excretory and secretory products of developing larvae on monophenol oxidase activity are suggested.

Demonstration of Monophenoloxidase Activity of Tyrosinase after Electrophoresis

The improved method presented here for localizing monophenoloxidase activity of tyrosinase (E.C. 1.14.18.1) after electrophoresis is based on the transfer of electrons from the monophenolic substrate, tyrosine methyl ester, to an artificial acceptor, phenazine methosulfate, and subsequent reduction of nitro blue tetrazolium into a violet formazan. This method is rapid, sensitive and versatile compared to the standard method. The electron transferred from monophenol can be accepted directly by nitro blue tetrazolium; although the background of the gel is clear, the sensitivity is decreased. The monophenol-PMS-NBT method is suitable for both plant and animal samples. This method can also be used for histochemical demonstration of monophenoloxidase activity.

Parasite‐induced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster

Larval hemolymph tyrosinase activity in Drosophila melanogaster was detected with high performance liquid chromatography with electrochemical detection. The enzyme hydroxylated L-tyrosine, and oxidized the diphenol substrates L-dopa and dopamine. In larvae of a selected immune-reactive strain the rates of tyrosine hydroxylation, dopa oxidation, and dopamine oxidation were markedly increased during the early stages of melanotic encapsulation of the eggs of the parasitic wasp Leptopilina boulardi. Tyrosinase activity was not modified in parasitized larvae of a selected susceptible strain of D. melanogaster, in which hosts the parasitoids developed unmolested. During the same period of parasitization, the amount of free tyrosine in immune reactive larvae was approximately three times higher than in susceptible hosts. These data indicate that the tyrosinase system of the immune reactive strain is activated during parasitization, and this results in the synthesis of some precursors which ultimately produce a melanotic and sclerotic capsule around the eggs of the parasite. Based on known genetic information of the enzyme system in Drosophila, it appears that at least two genes may be involved in the activation process, one associated with the proenzyme for monophenol oxidase activity, and the second with the proenzyme for diphenol oxidase activity.

Effect of γ irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

The effect of γ irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15–20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15–20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External γ irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in γ-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.

Host hemolymph monophenoloxidase activity in parasitized Manduca sexta larvae and evidence for inhibition by wasp polydnavirus

In insects, one of the primary routes of defense against parasites is encapsulation by hemocytes followed by melanization, in which tyrosine and DOPA are converted to melanin via toxic quinone intermediates. This report describes the use of an in vitro radiochemical assay to monitor hemolymph monophenoloxidase (MPO) conversion of [ 3H]tyrosine to [ 3H]DOPA, using a method utilized previously for dipteran and mammalian enzymes. Parasitism of fifth instar tobacco hornworm larvae by the braconid wasp Cotesia congregata depresses the rate of hemolymph monophenoloxidase activity in the host. Significant inhibition of hemolymph MPO was detectable in newly parasitized larvae and terminal stage hosts. A similar effect was seen in unparasitized larvae following injection of sucrose-gradient purified wasp polydnavirus (PDV) particles, which are normally injected by the female wasps into the host, suggesting the inhibition may be virally mediated. Hemolymph MPO activity was assayed in vitro 24 h after injection of PDV in vivo, and intercalation of viral DNA by exposure to psoralen and long-wave u.v. light eliminated its inhibitory effect on MPO. Despite inhibition of hemolymph MPO activity by parasitism, host cuticular enzymes appear unimpaired, since cuticular melanization occurs at sites of integumental wounding during emergence of the wasps from the host. Red pigments appear in the dorsal vessel and integument of parasitized and virus-injected larvae; whether these pigmentation changes are related to effects of parasitism on tyrosine metabolism and ommochrome biosynthesis remains to be determined.

Hemocyte Monophenol Oxidase Activity in Mosquitoes Exposed to Microfilariae of Dirofilaria immitis

Monophenol oxidase (MPO) activity in hemocytes collected from Aedes aegypti Liverpool strain and Aedes trivittatus intrathoracically inoculated with saline alone, inoculated with Dirofilaria immitis microfilariae (mff), or from uninoculated mosquitoes was compared using a radiometric tyrosine hydroxylation assay. Hemocyte MPO activity in mff-inoculated (= immune-activated) mosquitoes was significantly increased at 24 hr postinoculation (PI) in A. aegypti and at 6, 12, and 24 hr PI in A. trivittatus as compared with saline-inoculated controls. Baseline and immune-activated levels of hemocyte MPO activity in A. trivittatus were significantly higher compared with those seen in A. aegypti. Baseline hemocyte population levels were similar in both species, but immune activation did not elicit increases in total hemocyte populations in A. trivittatus as has been demonstrated for A. aegypti. Likewise, immune activation by the inoculation of mff did not significantly alter plasma MPO activity in A. trivittatus as compared with uninoculated or saline-inoculated mosquitoes. Plasma MPO activity in A. aegypti, however, appears to constitute a major component of the immune response. The importance of phenol oxidase(s) in the immune response of mosquitoes against mff and the relationship of observed differences in MPO activity to differences in immunological capability between A. aegypti and A. trivittatus are assessed.

Phenol oxidase activity during melanotic tumor formation in Drosophila melanogaster

Radiometric and spectrophotometric studies were made of larval phenol oxidase activity in the sex-linked recessive, temperature-sensitive, lethal mutant Tum 1 (1, 34.5) of Drosophila melanogaster during formation of melanotic tumors. At the tumor-expressive temperature of 29°C, virtually no phenol oxidase (monophenol or diphenol oxidase) was detected in either the hemolymph or hemocytes of hemizygous (male) Tum 1 larvae during early (72 hr) and late stages (96 and 120 hr) of melanotic tumor formation. Compared to normal Basc, heterozygous (female) Tum 1 showed reduced hemolymph and hemocyte enzyme activities during the two developmental stages studied (96 and 120 hr). At 120 hr, when melanotic tumors were completely manifested in female Tum 1, total hemolymph monophenol oxidase activity in this mutant was 50% that of Basc and hemolymph diphenol oxidase activity approximately 40% of the activity of Basc hemolymph with three enzyme substrates tested (dopa, dopamine, and N-acetyldopamine). Hemocyte diphenol oxidase activity in female Tum 1 at this stage of development also was comparably reduced to approximately intermediate levels (59% of the activity found in Basc cells), but hemocyte monophenol oxidase activity was substantially lower than the controls (25% of the activity of Basc). These results suggest that monophenol and diphenol oxidase activities in the Tum 1 mutant are regulated either by the mutant locus itself, or by linked modifiers also on the X chromosome. The progressive reduction in enzyme activity during melanotic tumor formation may result from product inhibition, the sequestering of enzyme-melanoprotein complexes within the pigmented masses, or the depletion of requisite enzyme activators or substrates.

Quantitative analysis of hemolymph monophenol oxidase activity in immune reactive Aedes aegypti

A hemocyte-mediated melanotic encapsulation reaction is elicited in adult Aedes aegypti in response to intrathoracically inoculated microfilariae of Dirofilaria immitis. The activity of monophenol oxidase in cell-free hemolymph collected from uninoculated, microfilariae-inoculated and saline-inoculated control mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of l-[3,5- 3H]tyrosine to dopa. Enzyme activity in immune reactive hosts examined 2 days postinoculation was aproximately twice as high (96–206 nmol/min per mg protein) as in uninoculated or saline inoculated insects (34–80 nmol/min per mg protein). The augmented activity of the enzyme coincides in time with the early development of melanotic capsules around the microfilariae. The possible involvement of hemocytes in the activation and/or generation of monophenol oxidase in response to infection, and the metabolism of catecholamines in relation to insect immune responses are discussed.