Phenoloxidase Inhibitors Research Articles

Ultrastructural and functional maturation of teratocytes of Apanteles kariyai

AbstractEggs and larvae of Apanteles kariyai avoid the host defense reactions of Pseudaletia separata due to the action of calyx and venom fluids injected by females during oviposition and the teratocytes originated from the embryonic serosal cells 3.5 day postoviposition. Phenoloxidase (PO) activity in host larvae was unaffected during early stages of parasitization (4–6 days postoviposition), relative to unparasitized larvae, but was greatly reduced to 25% during the late stage of parasitization (days 7–10). Hemolymph PO activity was not affected, in vitro, by calyx and venom fluids but was reduced in the presence of teratocytes. An apparent PO inhibitor was detected in older teratocyte cells. First instar parasitoid larvae implanted into unparasitized hosts, following transfer of either young teratocytes (4 day postoviposition) or old teratocytes (8 day post‐oviposition) with calyx and venom fluids resulted in avoidance of encapsulation only when calyx and venom fluids with young (4 day) teratocytes were injected. These results indicated that during early parasitization of the host, teratocytes just released from the embryonic serosal cells (4 day) function in conjunction with calyx and venom fluids injected into the host with the parasitoid egg to prevent its encapsulation by host hemocytes. During late parasitization, the older teratocytes (8 day) may also function in preventing host encapsulation by producing an PO inhibitor suppresses host hemolymph PO activity at the time of parasitoid egression.

Phenol-sensitive enzyme electrode with substrate cycling for quantification of certain inhibitory aromatic acids and thio compounds

A Clark oxygen electrode coated with a membrane of crosslinked champignon phenol oxidase (tyrosinase)is able to detect phenol containing analytical samples 3-5 times more sensitively after the admixture of hydrazine hydrochloride to the reaction buffer. This compound will recycle the substrate via chemical reduction of the quinoid product thus simultaneously preventing the membrane from rapid blackening. The decrease of oxygen in the membrane due to phenol oxidation is rapidly compensated by diffusion from the bulk solution when an inhibitor of the membrane-bound phenol oxidase is added to the reaction medium. This effect has been utilized for the measurement of low concentrations of compounds decreasing the sensitivity of the bioelectrode either reversibly (benzoic acid, 3- and 4-aminobenzoic acid, salicylic acid, 4-aminosalicylic acid, nicotinic acid, 4-nitrophenol, 1-naphthol) or irreversibly (phenylthiourea, thioacetamide, L-cysteine, reduced glutathione, 2-mercaptoethanol, 2,3-dimercaptopropanol and others). Benzoate as a potent reversible inhibitor markedly stabilizes the phenol oxidase reaction membrane during its storage either in the buffer or in dry state at 4 °C for one year at least.

On the mechanism of side chain oxidation of N‐β‐alanyldopamine by cuticular enzymes from Sarcophaga bullata

The metabolism of N-beta-alanyldopamine (NBAD) by Sarcophaga bullata was investigated. Incubation of NBAD with larval cuticular preparations resulted in the covalent bindings of NBAD to the cuticle and generation of N-beta-alanyl-norepinephrine (NBANE) as the soluble product. When the reaction was carried out in presence of a powerful quinone trap viz., N-acetylcysteine, NBANE formation was totally abolished; but a new compound characterized as NBAD-quinone-N-acetylcysteine adduct was generated. These results indicate that NBAD quinone is an obligatory intermediate for the biosynthesis of NBANE in sarcophagid cuticle. Accordingly, phenylthiourea--a well-known phenoloxidase inhibitor--completely inhibited the NBANE production even at 5 microM level. A soluble enzyme isolated from cuticle converted exogenously supplied NBAD quinone to NBANE. Chemical considerations indicated that the enzyme is an isomerase and is converting NBAD quinone to its quinone methide which was rapidly and nonenzymatically hydrated to form NBANE. Consistent with this hypothesis is the finding that NBAD quinone methide can be trapped as beta-methoxy NBAD by performing the enzymatic reaction in 10% methanol. Moreover, when the reaction was carried out in presence of kynurenine, two diastereoisomeric structures of papiliochrome II-(Nar-[alpha-3-aminopropionyl amino methyl-3,4-dihydroxybenzyl]-L-kynurenine) could be isolated as by-products, indicating that the further reactions of NBAD quinone methide with exogenously added nucleophiles are nonenzymatic and nonstereoselective. Based on these results, it is concluded that NBAD is metabolized via NBAD quinone and NBAD quinone methide by the action of phenoloxidase and quinone isomerase respectively. The resultant NBAD quinone methide, being highly reactive, undergoes nonenzymatic and nonstereoselective Michael-1,6-addition reaction with either water (to form NBANE) or other nucleophiles in cuticle to account for the proposed quinone methide sclerotization.

Tyrosinase-catalyzed unusual oxidative dimerization of 1,2-dehydro-N-acetyldopamine.

Tyrosinase, which usually catalyzes the conversion of o-diphenols to o-benzoquinones, catalyzed an unusual oxidative dimerization of 1,2-dehydro-N-acetyl-dopamine to a benzodioxan derivative. The identity of the product was confirmed by UV, IR spectra, and NMR studies. During the oxidation, generation of a transient reactive intermediate could be witnessed by its characteristic visible absorption spectrum. Typical phenoloxidase inhibitors such as phenylthiourea, potassium cyanide, sodium azide, and sodium fluoride drastically inhibited the above reaction. Mimosine, a known competitive inhibitor of o-diphenoloxidase activity, also inhibited the new reaction competitively, suggesting that both the observed oxidative dimerization and the conventional quinone production are catalyzed by the same active site copper of tyrosinase. Based on our earlier findings (Sugumaran, M., and Lipke, H. (1983) FEBS Lett. 155, 65-68; Sugumaran, M. (1986) Biochemistry 25, 4489-4492) that phenoloxidases can produce quinone methides from certain 4-alkylcatechols, possible mechanisms for this new reaction are presented.

In vitro study on humoral encapsulation of microfilariae: Effects of diethyldithiocarbamate and dopachrome on the reaction

The effects of a phenoloxidase inhibitor, diethyldithiocarbamate, and an intermediate of the melanin biosynthetic pathway, dopachrome, on the humoral encapsulation of microfilariae were studied in vitro. Diethyldithiocarbamate inhibited the melanization but did not prevent the humoral encapsulation of microfilariae. In the presence of diethyldithiocarbamate in the in vitro system, transparent material in the form of numerous granules were deposited on the surface of the microfilariae and coalesced to form a consolidated transparent capsule around microfilariae. The thickness of transparent capsule was significantly greater than that of normal melanotic capsule. The transparent capsule material, which was probably the precursor of the melanotic capsule, was demonstrated histochemically as a protein-carbohydrate complex. The ultrastructure of transparent capsule showed that the transparent capsule material was very different from that of melanotic capsule material, being more flocculent and less granular in appearance. When dopachrome was concurrently present with the inhibitor in the in vitro encapsulation system, it failed to restore normal melanotic encapsulation. The data presented lead to the following conclusions: 1. The melanin biosynthetic pathway in the humoral encapsulation of microfilariae is similar to that found in the synthesis of mammalian melanin. 2. The humoral encapsulation of the microfilariae in vitro is a two-step process. The protein-carbohydrate complex is deposited on the surface of microfilariae. Then the tyrosine group of the complex is catalysed by phenoloxidase to melanin.

Identification of Anthurium andraeanum Cultivars by Gel Electrophoresis

Abstract The use of gel electrophoresis of different isozyme systems was investigated as a possible tool to identify Anthurium andraeanum cultivars. Procedures were developed to extract active enzymes from anthurium leaves. Clear zymograms were obtained from only one of three extraction methods examined. This method consisted of grinding the leaf tissue in liquid nitrogen and then adding to the ground tissue a buffer containing a phenoloxidase inhibitor, reducing agents, and polyvinylpolypyrrolidone (PVPP). The identification of seven anthurium cultivars was undertaken using seven enzyme systems. Bands were observed in four of these systems; glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), peroxidase (Px), and phosphoglucose isomerase (PGI). All seven cultivars were characterized by the combined data of Px, MDH, and PGI.

Quelques aspects des interactions entre litières forestières et écosystèmes aquatiques ou terrestres

We have examined the processes responsible for the disturbing effects of dead leaves in freshwater environments. The first is a biochemical deoxygenation caused by the polyphenoloxidase-polyphenol system, which remains active in dead leaves. The intensity of this effect considerably varies from species to species. It depends on the quantity and nature of the phenolic substrates, as well as of the phenoloxidase activities, the presence of variable amounts of phenoloxidase inhibitors such as tanins, and the aqueous extractibility of these agents. The deoxygenating effect of dead leaves macerates is immediate and intense for some species, for example Poplar and Ash, but weak for others, such as Oak and Birch. This first process is followed by two others : 1) microbiological deoxygenation and 2) spontaneous precipitation of phytomelanins resulting from their oxidative polycondensation. These phytomelanins have ion exchange and sequestration properties. Nevertheless their disturbing effects on the aquatic environment are poorly understood at the present time. A fourth process is the toxicity of leaf macerates. This toxicity is probably due to their phenolic compounds, their oxidation products, or other compounds such as saponosides. Thus the Birch, in spite of a very weak deoxygenating effects is very toxic for juvenile roaches. We studied these four different processes in ten different species, and we established their respective role in the pollution of freshwater by dead leaves. These roles are discussed in terms of the phylogeny of the tree species studied, as well as of the functionning of temperate forest ecosystems.

Thiourea: The antioxidant of choice for the purification of proteins from phenol-rich plant tissues

Metabisulfite, diethyldithiocarbamate, and thiourea are potent phenoloxidase inhibitors commonly used during the extraction of plant proteins. Their effects on several amino acid derivatives and peptides are reported. Important and pH-dependent changes are induced by metabisulfite in the uv-absorption spectrum of N-acetyltryptophanamide but not of N-acetyltyrosinamide. Similar spectral changes are also induced in tryptophanyl-containing peptides. Neither diethyldithiocarbamate nor thiourea modifies the spectral properties of tryptophan or tyrosine. Cysteinyl groups are rapidly modified by diethyldithiocarbamate, especially in the lower pH range, but not by metabisulfite or thiourea. Diethyldithiocarbamate as well as metabisulfite interfere with cystinyl groups. It is concluded that thiourea is the most appropriate reagent for use as a phenoloxidase inhibitor.

Survival of endoparasitic Hymenoptera in pupae of Lepidoptera by inhibition of phenol oxidase

Die Larven der IchneumonidenPimpla turionellae undItoplectis conquisitor bewirkten in den Puppen der LepidopterenGalleria mellonella, Pieris brassicae undLymantria dispar eine verschieden hohe Melanisierung, die eine unterschiedliche Uberlebensrate der Larven zur Folge hatte: 1) Hohe Uberlebensrate vonP. turionellae in den WirtenG. mellonella undL. dispar. 2) Geringe Uberlebensrate der ParasitenP. turionellae inP. brassicae undI. conquisitor inG. mellonella. 3) Kein Uberleben vonI. conquisitor inP. brassicae. Das Ausmas der Melanisierung in den Organen der Wirts-Puppen wurde untersucht und die Auswirkung auf den Stoffwechsel von Wirt und Parasit diskutiert. Die Phenoloxidase-Aktivitat wurde in der Hamolymphe der drei Wirts-Arten gemessen sowie deren Hemmung durch das „Giftsekret” der adulten Parasitenweibchen und das „Analsekret” der Larven. Das Giftsekret zeigte eine lang anhaltende, schwache Hemmung der Phenoloxidase-Aktivitat, das Analsekret dagegen eine sehr starke, kurzzeitige Hemmung. Sowohl das Gift- als auch das Analsekret der einzelnen Parasiten hatte eine unterschiedliche Hemmwirkung auf die Phenoloxidase-Aktivitat der einzelnen Wirte, welche die unterschiedlichen Uberlebensraten in den drei Gruppen erklaren konnte.

Weitere Untersuchungen zur Beeinflussung der Phenoloxidase durch Effektoren

With the aid of the histochemical phenoloxidase (PO) assay, 12 substances were tested. On the one side they can act as substrates or as inhibitors of phenoloxidase and on the other side as immunogenic or immunosuppressive compounds. In particular the following substances were investigated: o-cresol, Levamisol, prednisolonebisuccinate, Bleocin, siliciumdioxide, 4-methylbrenzcatechol, +-catechine, thiomersal, D-penicillamine, hydrochinonemonomethylether, hydrochinonemonoethylether, and hydrochinonemonobenzylether. No changes in the phenoloxidase activity and in the morphological behaviour of the cells could be detected using Levamisol, prednisolonebisuccinate, siliciumdioxide, and Bleomycin, indicating an immunogenic stimulation or immunosuppressive effect. 4-methylbrenzcatechol is probably transformed into a soluble reaction product by phenoloxidase. It could not clearly demonstrated by the histochemical method, if this substance is used as a true substrate. +-catechine, thiomersal, and D-penicillamine cause strong to complete enzyme inhibition of the phenoloxidase containing cells (POC), which are located in the spleen and gut of the white rat. Hydrochinonemonoethylether, hydrochinonemethylether, and hydrochinonemonobenzylether cause an increased pigment fermentation, but they are not used as substrates for the PO.

Schistosoma mansoni: Phenol oxidase's role in eggshell formation

Phenol oxidase may be involved in the formation of the eggshell in Schistosoma mansoni. 3H-Labeled female S. mansoni proteins were polymerized in vitro following incubation with S. mansoni phenol oxidase and excess l-tyrosine. Peroxidase inhibitors, autoxidation inhibitors, inhibitors of lipid peroxidation, and inactive analogs of phenol oxidase inhibitors did not inhibit eggshell formation. Fluorescent substances found in eggshell hydrolysates were similar to those formed from the reaction of phenol oxidase-generated quinones with protein-bound lysine. These observations support the classical concept of phenol oxidase-catalyzed protein hardening. However, fluorescent globules of egg material were still formed after the administration of 200 mg/kg of the phenol oxidase inhibitor diethyldithiocarbamate. These globules could not be destroyed by inhibitors of autoxidation and lipid peroxidation.

The Effect of Mercaptoethanol on the Activity of Enzymes of Nitrogen Metabolism in Leaves from Urtica dioica and Spinacia oleracea

AbstractThe effect of mercaptoethanol at different concentrations on enzyme activity was investigated in leaves from Urtica dioica and Spinacia oleracea. The interference of mercaptoethanol with enzyme activity is dependent on the type of plant, the configuration of the enzyme and the concentration of mercaptoethanol. A stimulation of GDH (glutamate‐dehydrogenase) was obtained in both species, while inhibition of GOT (glutamate‐oxaloacetate transaminase) and GPT (glutamate‐pyruvate transaminase) was demonstrated in Spinacia. The stimulation of GOT and GPT activity in Urtica was probably due to inhibition of phenol oxidase. Conclusions concerning the effect of mercaptoethanol on NR (nitrate reductase) activity were difficult to draw, since mercaptoethanol itself reduced nitrite and interfered with NR determination in tests in vitro. In Urtica. no activity could be obtained at all with the in vitro method, probably because an inhibitor of NR was liberated during the extraction procedure, Activity of NR could however be obtained in both species when using the in vivo method. Addition of protective agents to the extraction medium has been supposed to influence the protein extractability. In conformity with this increasing amount of fresh matter to the same volume of extraction medium resulted in decreased protein extractability. This led to differences in enzyme activity when expressed on a fresh weight basis but the specific activity remained constant.

Phenoloxidase activity in the haemolymph of parasitized and unparasitized Heliothis virescens

Hymenopterous parasitoids of Heliothis virescens are able to evade the immune mechanisms of their host, especially the ability of the host to encapsulate foreign objects within a haemocyte capsule. This encapsulation is often accompanied by melanization. Our present findings show that hosts parasitized by Microplitis croceipes, Cardiochiles nigriceps and Campoletis sonorensis show changes both in the amount of melanin formed when their haemolymph is exposed to air and in the total protein as determined by the Folin phenol reagent. Furthermore, these changes are promoted by each of the three parasitoids in a different way. However, none of these parasitoids was able to alter the levels of host phenoloxidase as measured by the ability of the haemolymph to convert 4-methylcatechol to its quinone. The hymenopteran egg must therefore escape encapsulation in Heliothis in a way other than through inhibition of phenoloxidase, since the melanization reactions in the host's haemolymph are apparently unimpaired, particularly during the egg stage, by either the egg or venom of any one of the three parasitoids.

Protein Synthesis in Plant Microsomal System

AbstractAn active microsomal system from 48‐h germinating seeds of Vigna sinensis (L.) Savi has now been developed. It can incorporate amino acids into protein under both in vitro and in vivo conditions, provided dithiothreitol (a protective reagent for SH groups) and phenylthiourea (an inhibitor of phenol oxidase) are present in the buffer system for extraction; and provided the assay mixture contains added dithiothreitol.The system consists of microsomes or ribosomes, tRNA or pH 5 fraction and 20 natural amino acids, ATP and an ATP‐generating system and GTP with requirement for Mg ions. The cell fractions possess aminoacyl‐RNA synthetase activity as indicated by the aminoacylhydroxamate formation. Microsomal synthesis is stimulated by exogenous tRNA from Escheriehia coli or rat liver and sensitive to various inhibitors such as cyclo‐heximide, chloramphenicol, fusidic acid. The ribosomal transfer reaction has absolute dependence on the microsomal wash, on the crude enzyme from the same participate source, and on a synthetic messenger. It is greatly suppressed by fusidic acid and by cycloheximide.

Role of Sclerin on Morphogenesis inSclerotinia sclerotiorumde Bary (includingS. libertianaFuckel)

A correlation existed between the productivity of sclerin (SCL) and formation of sclerotia or dark pigmentation of old hyphae in Sclerotinia libertiana Fuckel and S. sclerotiorum de Bary. SCL stimulated the activities of phenoloxidase and peroxidase in a mycelial extract of S. libertiana, which appeared to be involved in the maturation and pigmentation of sclerotia. On the other hand, p-aminobenzoic acid (PABA), an inhibitor of phenoloxidase, inhibited the production of both SCL and sclerotia, where assimilation of PABA probably interrupted the biosynthetic process of SCL. When SCL was added at a high concentration, 4×10−3m, to Czapek’s agar medium a strain of S. libertiana which originally formed sclerotia and a strain of S. sclerotiorum which did not form sclerotia but produced melanin-like pigments at the edge of colony formed numerous sclerotium initials, which did not develop normal sclerotia. It was suggested that SCL played a role on induction of sclerotial formation and melanogenesis in these strains, in which a regulation of carbohydrate metabolism was involved.

Role of sclerin on morphogenesis in Schlerotinia sclerotiorum de bary (including S. libertiana Fuckel).

A correlation existed between the productivity of sclerin (SCL) and formation of sclerotia or dark pigmentation of old hyphae in Sclerotinia libertiana Fuckel and S. sclerotiorum de Bary. SCL stimulated the activities of phenoloxidase and peroxidase in a mycelial extract of S. libertiana, which appeared to be involved in the maturation and pigmentation of sclerotia. On the other hand, p-aminobenzoic acid (PABA), an inhibitor of phenoloxidase, inhibited the production of both SCL and sclerotia, where assimilation of PABA probably interrupted the biosynthetic process of SCL. When SCL was added at a high concentration, 4×10−3m, to Czapek’s agar medium a strain of S. libertiana which originally formed sclerotia and a strain of S. sclerotiorum which did not form sclerotia but produced melanin-like pigments at the edge of colony formed numerous sclerotium initials, which did not develop normal sclerotia. It was suggested that SCL played a role on induction of sclerotial formation and melanogenesis in these stra...

The toxic effects of phenoloxidase from the haemolymph of tenebrionid beetles

The injection of haemolymph originating from several species of tenebrionid beetles into blowfly larvae caused a gradual paralysis accompanied by colour changes in the haemolymph of the injected test insects. It was found that the lethal effect of the haemolymph of the beetle Blaps sulcata was due to phenoloxidase. The enzyme was activated by the exposure and incubation of the haemolymph at room temperature. The identity between the toxic factor and phenoloxidase in the beetle's haemolymph was demonstrated by the following data: (1) A correlation between the rate of lethal and phenoloxidase activities during the activation process of the toxic haemolymph. (2) Phenylthiourea, a well-known inhibitor of phenoloxidase, inhibited both the enzymatic and the toxic action of the beetle's haemolymph. (3) A commercial preparation of phenoloxidase (originating from mushrooms) imitated the lethal effects and the accompanying symptoms of the toxic haemolymph. (4) Sephadex G-100 column separation of the Blaps haemolymph revealed a complete overlap between the enzymatic and lethal regions of the elution pattern. The possible effects of phenoloxidase on the haemolymph of the injected insects are discussed.

Effects of some phenoloxidase inhibitors on chloroplasts and carboxylating enzymes of sugar cane and spinach

A number of additives have been tested for their effects on o-diphenol: O2 oxidoreductase activity of cane leaves. The most inhibitory compounds were thioglycollate, β-mercaptoethanol, polyethylene glycol and bovine serum albumin. Sulphydryl (SH) compounds did not affect rates of photosynthetic CO2 assimilation when used at concentrations below 10(-2) M. However, in the presence of Mn(++) ions they contributed to an O2 consumption which masked photosynthetic O2 evolution. Addition of SH compounds or of polymers to the grinding media increased rates of enzymic CO2 assimilation in crude enzyme preparations from cane leaves, but did not affect rates of CO2 assimilation in similar spinach preparations. Strong reducing agents, copper chelators, low O2 tension and high pH were effective in reducing phenoloxidase activity, but presented problems in the isolation and assay of chloroplasts. The results are discussed in relation to (a) design of suitable media for preparation of active cane chloroplasts and (b) comparative studies of enzyme levels in plants of various genera.

Effect of inhibitors on phenoloxidase of Mycobacterium leprae.

Previous results had shown that the human leprosy bacilli possess a phenoloxidase, which, when compared with the enzyme from mammalian and plant sources, seemed unique in the range of substrates utilized and in the nature of the products formed. The effect of several inhibitors on the enzyme in Mycobacterium leprae was tested. Compounds which bind copper were found to be more effective than substrate analogues. Diethyldithiocarbamate penetrated the bacillus and completely suppressed its phenolase activity. Diasone (a derivative of diaminodiphenylsulfone used in the treatment of leprosy) proved to be a potent inhibitor of phenoloxidase of mammalian and plant origin. However, it was less efficient in the case of M. leprae. A biochemical peculiarity of M. leprae was observed in its ability to metabolize mimosine and penicillamine. These compounds produced total inhibition of tyrosinase in melanoma extract and of mushroom tyrosinase. Nontoxic inhibitors of phenoloxidase in the leprosy bacilli may be of value in developing a rational approach to chemotherapy of the disease.

Oxidation of phenolic compounds by Mycobacterium leprae and inhibition of phenolase by substrate analogues and copper chelators.

Experiments were conducted on the substrate specificity of phenoloxidase in Mycobacterium leprae, by using various phenolic compounds. Comparative studies were carried out with the enzyme from mammalian and plant sources. The phenolase of M. leprae was found to be similar to the enzyme of plant origin in oxidizing a variety of substrates; it was different from the mammalian enzyme, which has a limited substrate specificity. The findings confirmed that phenoloxidase is a specific property of M. leprae and is not a result of adsorption of host-tissue enzymes. The method used in separation of bacilli from infected tissues was evaluated for its effect on the viability of the organisms. This was tested by using M. lepraemurium as a model. The preparative procedure was found to have no adverse effect on the ability of the organisms to multiply in the mouse foot-pad. Several inhibitors of phenoloxidase have been tested-both substrate analogues and compounds which bind copper in the enzyme. Substances binding copper were found to be more effective. Since phenolase has been found to be a characteristic metabolic activity in M. leprae, nontoxic inhibitors of the enzyme offer possibilities of developing a rational chemotherapy of leprosy.