Specificity of the enzyme phenoloxidase and possible metabolic pathway of sclerotization in Paraplerurus sauridae

Activity of phenoloxidase in Paraplerurus sauridae on various substrates has been studied spectrophotometrically. The enzyme was shown to be nonspecific and capable of oxidizing both mono- and diphenols. It showed greater activity to some of the mono- and diphenols. Substrates with acidic side chain were oxidized less effectively. Based on the activity of the enzyme on different substrates, a possible metabolic pathway of sclerotization has been suggested.     

Soluble cuticular phenoloxidase in the gypsy moth Lymantria dispar

The soluble enzyme phenoloxidase (tyrosinase) from the larval cuticle of Lymantria dispar has been partially purified using Ultrogel ACA 34, and the activity has been determined using phenolic substrates. The enzyme exhibited more activity toward O-diphenolic substrates and monophenolic substrates. The enzyme is inhibited by diethyl dithiocarbamate, phenylthiourea, and thiourea. The enzyme has been localized in the 7% slab and disc PAGE as an intense band. The enzyme is suggested to be involved in wound healing. © 1992 Wiley-Liss, Inc.     

An enzyme from lupin seeds forming alanine derivatives of cytokinins

A new enzyme, which catalyses the conversion of the cytokinin zeatin to the alanine conjugate lupinic acid, has been partly purified from developing lupin seed (Lupinus luteus). Paired-ion, reverse phase HPLC was adapted to analyse the enzyme reaction quantitatively. The enzyme used O-acetyl-l-serine as the source of the amino acid residue, and it interacted with substrates in a ping pong bi bi mechanism. A number of adenine derivatives served as substrates, but preference was shown for compounds with high cytokinin activity. The possible role of the enzyme, tentatively called β-(9-cytokinin)alanine synthase or lupinic acid synthase, in the regulation of hormone activity is discussed.     

Control mechanisms of tanning in Corcyra cephalonica

Localisation of a prophenoloxidase in the cytoplasm of plasmatocytes was histochemically demonstrable in Corcyra cephalonica, by incubating the blood with different phenolic substrates. The activation of this latent enzyme was found to be controlled by a protein factor present in the cuticle. The haemolymph prophenoloxidase could be activated in vitro by prior incubation of the blood sample at 0°C. The treatment with NaCl, EDTA, or detergents did not cause any activation. The exposure of blood samples to gamma-rays, repeated freezing and thawing, or addition of the cuticular activator, brought about rapid disruption of the haemocytes causing a release of phenoloxidase and acceleration of melanization of the blood. The administration of α-ecdysone to the last instar larvae induced premature pupation and accentuated the activation of phenoloxidase without altering the level of enzyme activity. The possible regulatory mechanisms of tanning during the development of Corcyra have been discussed.     

Ultrastructural localization of phenoloxidase in the midgut of refractory Anopheles gambiae and association of the enzyme with encapsulated Plasmodium cynomolgi

A melanogenic enzyme, phenoloxidase, was localized ultrastructurally in the midgut epithelia of 2 strains of Anopheles gambiae, a refractory strain that melanotically encapsulates Plasmodium cynomolgi ookinetes on the midgut, and a susceptible strain that does not. Midguts were incubated with either dopa or dopamine, and the resultant electron-dense product of phenoloxidase activity was localized on the basal lamina (BL) and cellular basal membrane labyrinth (BML) in uninfected mosquitoes of both strains. In infected refractory mosquitoes, the reaction products still were observed on the BL and BML but were especially dense in the BML of midgut cells near encapsulated ookinetes and in the capsule itself. In infected susceptible mosquitoes, phenoloxidase localization was reduced or absent in the BL and BML and was not observed near parasites. Phenylthiourea (PTU) inhibited the phenoloxidase reaction, indicating that the reaction product deposited in the absence of PTU resulted from enzyme activity and not autooxidation of the substrates. It is concluded that higher levels of phenoloxidase in the refractory strain following a blood meal may contribute to the ability to encapsulate ookinetes.     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. I. Activation reaction by the enzyme

Prephenoloxidase-activating enzyme from larval cuticle of the silkworm catalyzes the activation reaction of hemolymphal prephenoloxidase to the active enzyme. The activation reaction of prephenoloxidase by the enzyme has been analyzed with respect to effect of salts, dependency on pH and substrate concentration, and susceptibility to inhibitors. It has been demonstrated that the reaction is highly sensitive to specific inhibitors for “serine enzyme.”Difference in substrate specificity of phenoloxidase preparations, produced by two enzyme fractions which can be separated by chromatography on DEAE-cellulose, is also described.     

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.     

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.     

Sensitive enzyme assays based on the production of chemiluminescent leaving groups

A new type of synthetic peptide substrate for amidase assay has been devised. The substrates are luminogenic, with potential for extremely high sensitivity, and are here exemplified by Boc- and Z-Ala-Ala-Phe-isoluminol amide. The synthetic substrates were designed to release isoluminol when hydrolyzed by enzyme; isoluminol production was determined by measuring its chemiluminescence. Kinetic constants of the luminogenic substrates were measured with α-chymotrypsin; and levels of the enzyme as low as 50 ng were determined conveniently. A comparison of similar luminogenic, chromogenic, and fluorogenic substrates is presented.     

Purification and characterization of a prophenoloxidase activating enzyme from crayfish blood cells

A proteinase was purified from crayfish haemocytes by affinity chromatography on heparin-sepharose and phenyl-sepharose, followed by DEAE-cellulose ion-exchange chromatography. This proteinase could mediate the conversion of prophenoloxidase (proPO) to its active form, phenoloxidase (PO), and its was therefore designated a prophenoloxidase activating enzyme, ppA.The purified ppA had a molecular mass of about 36,000 Da. Since ppA was a proteinase able to cleave chromogenic peptide substrates of trypsin, and serine proteinase inhibitors were strongly inhibitory towards ppA activity, the enzyme appeared to be a serine type proteinase. It exhibited maximal enzyme activity at neutral and slightly alkaline pH, and was sensitive to heat inactivation at 58°C.     

An aminopeptidase from Agave americana,chemical properties of the enzyme

Agave aminopeptidase, a new enzyme obtained from the plant Agave americana displayed activity towards a variety of substrates. A free alphaamino group on these substrates was essential, but the enzyme did not need any metal ions for optimal activity. Aliphatic, aromatic and basic amino acids situated at the amino terminal end of substrates could be hydrolysed by the enzyme. The enzyme had no endopeptidase or other proteolytic activity. Values of the apparent Michaelis constants for different amino acid substrates, all in the range from 0.1 to 0.6 × 10^?3 M, suggested a relative wide specificity. The pK-values of the two dissociating groups on the enzyme taking part in the catalytic process were pH 6.3 to 6.8 and pH 7.5 to 7.8. These and other studies suggested that histidine plays an active role in the catalytic process. The enzyme was inhibited competitively by free amino acids and this, together with other results, implied a compulsory order of product release.     

Attachment of phenoloxidase to fungal cell walls in arthropod immunity

In crayfish, phenoloxidase was located in the hemocytes. The plasma had infinitesimal enzyme activity. A phenoloxidase preparation from hemocytes precipitated spontaneously after approximately 1.5 hr at 22°C, which became attached spontaneously to glass, Plexiglas, and polystyrene plastic. The enzyme preparation could also become attached to Saccharomyces cerevisiae cell walls. Attachment was mediated by a proteinaceous substance, since trypsin significantly decreased the degree of attachment. Calcium ions were also necessary for attachment. A β-1,3-glucan, laminaran, partially prevented attachment to the fungal cell walls. Heparin caused precipitation of the phenoloxidase preparation from hemocytes. In crayfish cuticle, proteins with associated phenoloxidase activity were attached to cell walls of Aphanomyces astaci as well as to those of S. cerevisiae.     

Breeding for QX disease resistance negatively selects one form of the defensive enzyme, phenoloxidase, in Sydney rock oysters

QX disease in Sydney rock oysters (Saccostrea glomerata) is caused by the paramyxean protozoan, Marteilia sydneyi. Disease outbreaks occur during summer (January to May) and can result in up to 95% mortality. The New South Wales Department of Primary Industries has been selectively breeding S. glomerata for resistance to QX disease since 1996. Previous work suggests that this breeding program has specifically affected the defensive phenoloxidase enzyme system of oysters. The current study more thoroughly characterises the effect of selection on the different forms of phenoloxidase found in oyster populations. Native polyacrylamide gel electrophoresis (native-PAGE) identified five discrete types of phenoloxidase in non-selected (wild type) and fourth generation QX disease resistant (QXR4) oysters. One electrophoretically distinct form of phenoloxidase, POb, is significantly less frequent in resistant oysters when compared to the wild type population. The frequency of POb also decreased in both the wild type and QXR4 populations over the course of a QX disease outbreak. This suggests that possession of POb makes oysters susceptible to QX disease and that breeding for resistance has resulted in negative selection against this form of phenoloxidase.     

Purification of the prophenoloxidase from Hyalophora cecropia and four proteins involved in its activation

Prophenoloxidase (PPO) has been purified to homogeniety from hemolymph of Hyalophora cecropia. There are two forms of the enzyme with identical molecular weights (76 kDa). Four proteins directly involved in the activation of PPO have also been purified from the hemolymph. Active phenoloxidase is elicited by the addition of factor C1 and a serine protease (SPII), which alone cannot activate PPO. Purified SPII contains two proteins with M_r 43 and 53 kDa, the larger molecule may represent the unactivated enzyme. An inhibitor of the SPII catalyzed activation of PPO has been isolated. In addition a protein presumed to be dopa quinone imine conversion factor has been purified.     

Phenoloxidase activity and its rôle in cuticular sclerotization in a mole crab Emerita asiatica Milne Edwards

Phenoloxidase has been localized in the epicuticle, exocuticle, and epidermal cells of the mole crab, Emerita asiatica Milne Edwards. The enzyme activity in different moulting stages is in the order of freshmoult > premoult > intermoult = postmoult. The phenoloxidase of the freshmoult cuticle oxidizes pyrogallol and epinephrine (adrenaline) more effectively than the other phenols studied. There is no monophenolase activity. The possible metabolic pathway has been suggested based on the specificity of the enzyme and the chromatographic identification of the extracted phenols. Phenoloxidase shows different pH optima in different buffers. The protein pattern in the various moulting stages of the cuticle differs and the results are discussed in relation to sclerotization.     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. II. Purification and characterization of the enzyme

Prephenoloxidase-activating enzyme has been purified approximately 4800-fold from cuticular extract of the silkworm, and the preparation seems to be homogeneous as judged by disc- and dodecylsulfate-polyacrylamide gel electrophoresis. By means of gel filtration through Sephadex G-100, it has been supposed that the enzyme exists as mono- and dimeric forms at slightly acidic pH, while a monomeric form is predominant under slightly alkaline condition. The molecular weight of the monomer was estimated to be 33,000–35,000 by dodecylsulfate-polyacrylamide gel electrophoresis and gel filtration.It has been demonstrated that ester substrates for trypsin, benzoyl-l-arginine ethyl ester and tosyl-l-arginine methyl ester, can be hydrolyzed by the purified enzyme. Several lines of evidence indicating that a single protein is involved in both activation and esterolytic reactions have been presented. Some enzymatic properties of the purified preparation as esterase have also been described.In connection to esterase activity of the purified enzyme, a mechanism of prephenoloxidase activation in the silkworm system has briefly been discussed.     

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn²⁺ and Mg²⁺ and inactivation by Co²⁺ and Zn²⁺ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. K_m values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.     

Fungi from koala (Phascolarctos cinereus) faeces exhibit a broad range of enzyme activities against recalcitrant substrates

Aims:  Identification of fungi isolated from koala faeces and screening for their enzyme activities of biotechnological interest.
Methods and Results:  Thirty-seven fungal strains were isolated from koala faeces and identified by the amplification and direct sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA. The fungi were screened for selected enzyme activities using agar plates containing a single substrate for each target class of enzyme. For xylanase, endoglucanase, ligninase (ligninolytic phenoloxidase) and protease over two-thirds of the isolates produced a clearing halo at 25°C, indicating the secretion of active enzyme by the fungus, and one-third produced a halo indicating amylase, mannanase and tannase activity. Some isolates were also able to degrade crystalline cellulose and others displayed lipase activity. Many of the fungal isolates also produced active enzymes at 15°C and some at 39°C.
Conclusions:  Koala faeces, consisting of highly lignified fibre, undigested cellulose and phenolics, are a novel source of fungi with high and diverse enzyme activities capable of breaking down recalcitrant substrates.
Significance and Impact of the Study:  To our knowledge, this is the first time fungi from koala faeces have been identified using ITS sequencing and screened for their enzyme activities.     

A laccase-like phenoloxidase is correlated with lignin biosynthesis in Zinnia elegans stem tissues

Stem tissues from different internodes of 4–6 week-old Zinnia elegans cv. Envy plants were sectioned and stained with chromogenic substrates previously used in studies of laccases (p-diphenol:O₂ oxidoreductases) isolated from tree tissues. The pattern of color development found when stem sections were stained in the presence and absence of H₂O₂ suggested that p-diphenol:O₂ oxidoreductase activity was tightly correlated spatially and temporally with the lignification of secondary cell walls in developing primary xylem. The correlation between this laccase-like phenoloxidase activity and lignification appeared tighter than that between lignification and peroxidases stained using the same substrates. Zymogram analysis of the phenoloxidase activities catalyzed by enzymes that were not boiled prior to separation by SDS—PAGE suggested that a single enzyme was predominantly responsible for the laccase-like phenoloxidase activity in Zinnia stems. Some of this enzyme was released from cell wall residue by washing with high ionic strength buffer; however, substantial amounts of the enzyme could only be recovered after treatment of the residue with cell wall-degrading enzymes. This phenoloxidase appears to share significant characteristics with the coniferyl alcohol oxidase isolated from developing secondary xylem in pines, which suggests that such enzymes may be widespread in vascular plants.     

Regulation of enzyme activity in adsorptive enzyme systems

The kinetic behaviour of adsorptive enzyme systems with free and adsorbed enzyme forms in rapid equilibrium has been analysed. It has been shown that the dependences of enzymic reaction rate on substrate or “adsorptive effector” concentrations reveal the deviations from simple kinetic laws of Michaelis-Menten type (positive or negative kinetic co-operativity). Such kinetic anomalies should be observed when adsorption of the enzyme results in the changing catalytic properties and when the state of the equilibrium between free and bound enzyme forms depends on the presence of low molecular substances (substrates, coenzymes and various cellular metabolites). The physiological significance of adsorption-desorption processes for the enzyme activity regulation has been emphasized.