Monophenolase activity of avocado polyphenol oxidase

Polyphenol oxidase of avocado mesocarp catalyses (a) the orthohydroxylation of monophenols like l-tyrosine, d-tyrosine, tyramine and p-cresol, and (b) the oxidation of the corresponding o-dihydroxyphenols to quinones. The rate of step b is much greater than that of step a. The hydroxylation of monophenols occurs after a lag period. DOPA or ascorbate effectively eliminate the lag but not dl-6-methyltetrahydropteridine or tetrahydrofolic acid. At 1.66 × 10^?4 M, α,α-dipyridyl has no effect, while diethyldithiocarbamate at this concentration inhibits the hydroxylation reaction by 90%. The tyrosinase activity of avocado polyphenol oxidase is inactivated in the course of the reaction; this inactivation occurs faster and is more pronounced in the presence of exogenously added DOPA. This inactivation is partially prevented by a large excess of ascorbate. The K_m values indicate that tyramine, dopamine, p-cresol and 4-methyl catechol are better substrates for avocado polyphenol oxidase than tyrosine or DOPA.     

Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.     

Decolorization and removal of textile and non-textile dyes from polluted wastewater and dyeing effluent by using potato (Solanum tuberosum) soluble and immobilized polyphenol oxidase

Celite bound potato polyphenol oxidase preparation was employed for the treatment of wastewater/dye effluent contaminated with reactive textile and non-textile dyes, Reactive Blue 4 and Reactive Orange 86. The maximum decolorization was found at pH 3.0 and 4.0 in case of Reactive Blue 4 and Reactive Orange 86, respectively. Immobilized potato polyphenol oxidase was significantly more effective in decolorizing the individual dye and complex mixtures of dyes as compared to soluble enzyme. The absorption spectra of the treated and untreated dye mixture and dyeing effluent exhibited a marked difference in the absorption value at various wavelengths. The polluted water contaminated with an individual dye or mixtures of dyes treated with soluble and immobilized potato polyphenol oxidase resulted in the remarkable loss in total organic carbon.     

Enhancement of In Vivo Tyrosine Hydroxylation in the Rat Adrenal Gland Under Hypoxic Conditions

We examined the effects of hypoxia (8% O2) on in vivo tyrosine hydroxylation, a rate-limiting step for catecholamine synthesis, in the rat adrenal gland. The hydroxylation rate was determined by measuring the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition. One hour after hypoxic exposure, DOPA accumulation decreased to 60% of control values, but within 2 h it doubled. At 2 h, the apparent Km values for tyrosine and for biopterin cofactor of tyrosine hydroxylase (TH) in the soluble fraction were unchanged, whereas the Vmax value increased by 30%. The content of total or reduced biopterin was unchanged, but the content of tyrosine increased by 80%. Tyrosine administration had little effect on DOPA accumulation under room air conditions but enhanced DOPA accumulation under hypoxia. After denervation of the adrenal gland, the hypoxia-induced increase in DOPA accumulation and in the Vmax value was abolished, whereas the hypoxia-induced increase in tyrosine content was persistent. These results suggest that in vivo tyrosine hydroxylation is enhanced under hypoxia, although availability of oxygen is reduced. The enhancement is the result of both an increase in tyrosine content coupled with increased sensitivity of TH to changes in tyrosine tissue content and of an increase in dependence of TH on tyrosine levels. The increase in the sensitivity of TH and in the Vmax value is neurally induced, whereas the increase in tyrosine content is regulated by a different mechanism.     

Polyphenol oxidase produced during encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a Km value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation, and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.     

Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION

The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H₂O as electron donor for the photoreduction of NADP, (b) NADP replaced O₂ as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O₂ at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.     

Polyphenol Oxidase Produced During Encystation of Acanthamoeba castellanii

Acanthamoeba castellanii has a phenol oxidase activity that is believed to be a laccase. Enzyme activity was found in the outer cyst wall, in the cytoplasm of encysting amoebae and in the encystment medium. Encystment procedures were modified to promote an increase in the amount of soluble enzyme secreted during encystation. Acanthamoeba polyphenol oxidase has a pH optimum of 6.0 and a K_m value of 0.21 mM with dihydroxyphenylalanine. The enzyme does not oxidize tyrosine, and it is inhibited by chloride but not by inhibitors of peroxidase. Its synthesis coincides with encystation. and known inhibitors of polyphenol oxidase prevent encystation. Polyphenol oxidase may have a role in making the cyst resistant to mechanical and chemical breakdown.     

Prevention of browning in potato with a heat-treated onion extract

The inhibitory effect of an onion extract on browning of potato was investigated. The addition of the heated onion extract to potato exhibited a marked inhibitory effect on potato polyphenol oxidase and the formation of a brown color. The inhibitory effect of the onion extract was dependent upon its heating temperature. The addition of both glycine and glucose increased the inhibitory effect of the onion extract toward potato polyphenol oxidase.     

Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris

The effect of regurgitant from Leptinotarsa decemlineata Say larvae on wound-induced responses was studied using two plant species, Solanum tuberosum L. and Phaseolus vulgaris L. Wounding of one leaf of intact S. tuberosum plants differentially affected ethylene production and activities of peroxidase and polyphenol oxidase. Only polyphenol oxidase activity was stimulated by wounding in both wounded and systemic leaves. Peroxidase activity was not affected by wounding. Wounding caused only a transient increase of ethylene production from wounded leaves. The application of regurgitant to wound surfaces stimulated ethylene production as well as activities of peroxidase and polyphenol oxidase in both wounded and systemic leaves. Wounding significantly enhanced ethylene production and polyphenol oxidase activity in wounded and systemic leaves of P. vulgaris . The application of regurgitant caused an amplification of ethylene production, peroxidase activity, and polyphenol oxidase activity, in both wounded and systemic leaves of bean plants. Several substances were tested for their role as possible endogenous signals in P. vulgaris . Hydrogen peroxide and methyl jasmonate appeared as potential local and systemic signals of ethylene formation in wounded bean plants. Local ethylene production in leaf discs was differentially affected by the regurgitant application in potato versus bean plants. While all tested concentrations of regurgitant caused stimulation of ethylene formation from potato leaf discs, ethylene production was completely inhibited by increasing concentrations of the regurgitant in bean leaf discs. Our data present evidence that ethylene may play an important role in the interaction between plants and herbivores at the level of recognition of a particular herbivore leading to specific induction of signalling cascades.     

Evidence against superoxide involvement in tyrosine hydroxylation by mushroom tyrosinase

The possible involvement of superoxide anions in the hydroxylation of tyrosine by mushroom tyrosinase was studied. Superoxide dismutase and scavengers of superoxide ions of smaller MW than superoxide dismutase, such as nitroblue tetrazolium and copper salicylate, had no direct effect on the monohydroxyphenolase activity of mushroom tyrosinase. The kinetics of tyrosine hydroxylation, but not of DOPA oxidation, by mushroom tyrosinase was atrected by the addition of a xanthine-xanthine oxidase system. In the presence of the xanthine-xanthine oxidase system, the lag period of tyrosine hydroxylation was shortened compared to the lag period in the absence of the xanthine-xanthine oxidase system. The xanthine- xanthine oxidase system alone (without mushroom tyrosinase) had no effect on tyrosine conversion to dopachrome. Superoxide dismutase, catalase and hydroxyl radical scavengers counteracted to some extent the shortening of the lag period of tyrosine hydroxylation by mushroom tyrosinase caused by the xanthin e-xanthine oxidase system. It is suggested that the shortening of the lag period is due mainly to hydroxyl radicals generated by the xanthine-xanthine oxidase system via interaction of O₂^?. and hydrogen paroxide (a Haber-Weiss type reaction). The data do not support the direct participation of superoxide anions in tyrosine hydroxylation by mushroom tyrosinase.     

Activity of Tyrosine Hydroxylase in the Striatum of Newborn Piglets in Response to Hypocapnic Hypoxia

Abstract: The effect of graded hypoxia induced by hyperventilation on the activity of tyrosine hydroxylase was measured in vivo by microdialysis. Microdialysis probes were inserted into the striatum of newborn piglets and perfused with medium containing 3-hydroxybenzylhydrazine, an inhibitor of L-aromatic amino acid decarboxylase. The level of 3,4-dihydroxyphenylalanine (DOPA) measured in the effluent dialysate was then an index of tyrosine hydroxylase activity. The oxygen pressure in the veins and capillaries of the cortex was measured, through a cranial window placed over the parietal cortex, by the phosphorescence lifetime of palladium-meso-tetra(4-carboxyphenyl)porphine added to the blood. After baseline measurements, P_aCO₂ was decreased from 38 torr (control value) to 19, 13, and 11 torr resulting in decreases in the cortical oxygen pressure from 40 ± 6 torr to 26 ± 3, 23 ± 4, and 20 ± 4 torr, respectively. Decrease in the oxygen pressure to 26 ± 3 torr caused a statistically significant increase of 25–30% in the level of DOPA in the effluent perfusate. During the next step of increase in ventilator rate, when oxygen decreased only slightly, the level of DOPA remained at the higher level. Ventilation rates that lowered the oxygen pressure to below 20 torr, however, caused a progressive decrease in the level of DOPA. During recovery, the level of DOPA steadily increased, attaining 160% of control value after 1.5 h. When the oxygen pressure was decreased to 16 ± 2 torr by a single increase in ventilator rate, the DOPA level decreased in the effluent to 15–20% below control. With return of the ventilator rate to control values, the DOPA levels again increased to well above control and stayed higher even after 1.5 h. The slow return of tyrosine hydroxylase activity to control indicates relatively long-term modification of the enzyme activity. Activation of tyrosine hydroxylase occurs when the oxygen pressure is decreased, but at <16 torr the reaction rate becomes limited by the availability of oxygen and decreases with further decrease in oxygen pressure. Our results showed that even small changes in cortical oxygen pressure modulate the activity of tyrosine hydroxylase. This alteration in the metabolism of catecholamines in newborn brain may have significant impact on later development of the organism.     

A STUDY ON INCREASE IN O-DIPHENOL OXIDASE ACTIVITY DURING INCUBATION OF SLICED SWEET POTATO TISSUE

The increase in o-diphenol oxidase activity and polyphenol contentwas investigated in slices excised from sweet potato roots.o-Diphenol oxidase activity increased in a sigmoidal fashionover a 100 hour period. The increase in polyphenols occurredover a shorter period of time and was evident before an increasein o-diphenol oxidase activity could be detected. Thus, it seemedthat the increase in polyphenol content might be involved inthe enhancement of o-diphenol oxidase activity. However, theabove correlation was not found in different kinds of experimentincluding pretreatment with either vacuum infiltration or wetconditioning. (Received October 14, 1965; )     

Tissue Browning of Potato Tubers Induced by Gamma Irradiation

A considerable browning was observed especially in cortex tissue and along xylem of potato tubers harvested at Sakai in Osaka Prefecture, after irradiation with 10, 20 and 50 krad doses of cobalt-60 gamma rays. This phenomenon was accompanied by the marked increase in polyphenol content and peroxidase activity, and the transient increase in o-diphenol oxidase activity. Total reducing compounds in the tissue were also increased by gamma irradiation.

The browning phenomenon depended on the storage period from the harvest to gamma irradiation treatment. The browning and the transient increase in o-diphenol oxidase activity were completely suppressed in the case of tubers irradiated 3 months after harvest.

There was no significant change in α-amylase activity in all tubers tested.     

Use of Microdialysis for Monitoring Tyrosine Hydroxylase Activity in the Brain of Conscious Rats

An on-line microdialysis system was developed which monitored the 3,4-dihydroxyphenylalanine (DOPA) formation in the striatum during infusion of a submicromolar concentration of an L-aromatic amino-acid decarboxylase inhibitor (NSD 1015). The absence of DOPA in dialysates of 6-hydroxydopamine-pretreated rats and the disappearance of DOPA after administration of alpha-methyl-p-tyrosine indicated that the dialyzed DOPA was derived from dopaminergic nerve terminals. Next we investigated whether the steady-state DOPA concentration in striatal dialysates could be considered as an index of tyrosine hydroxylase activity. The increase in DOPA output after intraperitoneal administration of haloperidol or gamma-butyrolactone and the decrease in DOPA output after intraperitoneal administration of apomorphine are in excellent agreement with results of postmortem studies, in which a decarboxylase inhibitor was used to measure the activity of tyrosine hydroxylase. The effect of haloperidol on DOPA formation was not visible when a U-shaped cannula (0.80 mm o.d.) was used. Some methodological problems related to microdialysis of the haloperidol-induced increase in DOPA formation are discussed. We concluded that the proposed model is a powerful and reliable in vivo method to monitor tyrosine hydroxylase activity in the brain. The method is of special interest for investigating the effect of compounds which are not able to pass the blood-brain barrier. As an application of the method in the latter situation, we report the effect of infusion the neurotoxin 1-methyl-4-phenylpyridinium ion (10 mmol/L infused over 20 min) on the activity of striatal tyrosine hydroxylase.     

Regulation of tyrosine hydroxylase activity and phosphorylation at Ser(19) and Ser(40) via activation of glutamate NMDA receptors in rat striatum

The activity of tyrosine hydroxylase, the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by phosphorylation. In this study, we examined the effects of activation of NMDA receptors on the state of phosphorylation and activity of tyrosine hydroxylase in rat striatal slices. NMDA produced a time-and concentration-dependent increase in the levels of phospho-Ser(19)-tyrosine hydroxylase in nigrostriatal nerve terminals. This increase was not associated with any changes in the basal activity of tyrosine hydroxylase, measured as DOPA accumulation. Forskolin, an activator of adenylyl cyclase, stimulated tyrosine hydroxylase phosphorylation at Ser(40) and caused a significant increase in DOPA accumulation. NMDA reduced forskolin-mediated increases in both Ser(40) phosphorylation and DOPA accumulation. In addition, NMDA reduced the increase in phospho-Ser(40)-tyrosine hydroxylase produced by okadaic acid, an inhibitor of protein phosphatase 1 and 2A, but not by a cyclic AMP analogue, 8-bromo-cyclic AMP. These results indicate that, in the striatum, glutamate decreases tyrosine hydroxylase phosphorylation at Ser(40) via activation of NMDA receptors by reducing cyclic AMP production. They also provide a mechanism for the demonstrated ability of NMDA to decrease tyrosine hydroxylase activity and dopamine synthesis.     

Effect of light and exogenously applied precursors on amaranthin synthesis in Amaranthus caudatus

Light stimulates the synthesis of amaranthin in Amaranthus caudatus var. viridis. Evidence suggests that this stimulation is markedly dependent on seedling age. Synthesis is controlled by both a “low-energy” red/far-red reversible phytochrome system and an HER at least partially under phytochrome control. In seedlings exposed to light, synthesis is promoted by exogenously applied DOPA and tyrosine. It is suggested that at least two light-promoted reactions occur in the biosynthetic pathway; one between tyrosine and DOPA and a second between DOPA and amaranthin.     

The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.     

Enhanced Tyrosine Hydroxylation in Hippocampus of Chronically Stressed Rats upon Exposure to a Novel Stressor

We have used microdialysis to measure the in vivo level of tyrosine hydroxylation in hippocampus of the freely moving rat. An inhibitor of aromatic amino acid decarboxylase, NSD-1015, was administered through the dialysis probe and the resulting accumulation of 3,4-dihydroxyphenylalanine (DOPA) in extracellular fluid of hippocampus was quantified. Administration of the tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, decreased extracellular DOPA to undetectable level. In addition, both systemic and local application of clonidine, an alpha 2-adrenergic agonist, produced a decrease in extracellular DOPA. In response to acute tail shock, a significant increase in extracellular DOPA was observed. Thus, it appears that in vivo accumulation of DOPA after local administration of NSD-1015 provides a reliable index of hippocampal tyrosine hydroxylation. We have used this technique to investigate whether prior exposure to chronic stress alters the in vivo level of tyrosine hydroxylation in hippocampus under basal conditions as well as in response to a novel stressor. In rats previously exposed to chronic cold stress, the basal accumulation of extracellular DOPA did not differ from naive controls. Acute tail shock, however, produced a significantly greater and more prolonged elevation in extracellular DOPA of chronically stressed rats. These data suggest that enhanced biosynthetic capacity of noradrenergic terminals may be one mechanism underlying adaptation to chronic stress.     

Changes in the location of polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue

In order to elucidate the nature of the response of potato to impact injury at the biochemical level, changes in the location of the enzyme responsible for the discoloration, polyphenol oxidase, were determined using immunogold location with an antibody specific for potato tuber polyphenol oxidase. Tissue printing revealed that the enzyme was distributed throughout the tuber. Following impact injury, both tissue printing and quantitative electron microscopy indicated that there was no increase in the level of the enzyme although there was subcellular redistribution of polyphenol oxidase. This redistribution was first apparent at 12 h after impact, as determined by the use of confocal immunolocation, and coincided with loss of membrane integrity. These changes were examined in parallel with a number of stress-related parameters in both impact and wound responses. Wounding was accompanied by active gene expression and protein synthesis, leading to metabolic activity and tissue repair. In contrast, the bruising response was characterised by a limited active response and vital-staining methods indicated that after 16 h the tissue undergoes cell death. Received: 4 June 1998 / Accepted: 18 September 1998     

Free and peptide-bound DOPA can inhibit initiation of low density lipoprotein oxidation

Hydroxyl radicals have been shown to convert free tyrosine to 3,4-dihydroxyphenyl-alanine (DOPA) which has reducing properties. During protein or peptide oxidation such reducing species are also formed from tyrosine residues. Free DOPA or peptide-bound DOPA (PB-DOPA) is able to promote radical-generating events, facilitating the damage of biomolecules such as nucleic acids. Radical induced lipid oxidation in low density lipoprotein (LDL) transforms the lipoprotein into an atherogenic particle. As PB-DOPA has been found in atherosclerotic plaques, we tested the ability of free and PB-DOPA to influence LDL oxidation. Free DOPA, in contrast to tyrosine had strong inhibitory action on both, the copper-ion initiated and metal ion independent (AAPH-induced) lipid oxidation. Free DOPA also inhibited LDL oxidation induced by the copper transport protein ceruloplasmin. To test if PB-DOPA was also able to inhibit LDL oxidation, DOPA residues were generated enzymatically in the model peptides insulin and tyr-tyr-tyr, respectively. PB-DOPA formation substantially increased the ability of both molecules to inhibit LDL oxidation by copper or AAPH. We hypothesize that DOPA-peptides and -proteins may have the potential to act as efficacious antioxidants in the atherosclerotic plaque.