Evidence for a common regulation in the activation of a polyphenol oxidase by trypsin and sodium dodecyl sulfate

Polyphenol oxidase (PPO) was extracted from beet root, in both soluble and membrane fractions, and in both cases the enzyme was in a latent state. PPO from the membrane fraction showed no diphenolase activity unless it was activated by trypsin or sodium dodecyl sulfate (SDS). The kinetics of the activation process of latent PPO by trypsin was studied and the specific rate constant of active PPO formation, k 3 , showed a value of 0.03 s(-1). The protease-activated form showed a pH optimum (6.5) and kinetic properties identical to those of the SDS-activated enzyme. Evidence is provided for the existence of a common peptide responsible for the regulation of the activity of the enzyme by both proteolysis and SDS detergent. Formation of the active proteolyzate was followed by spectroscopic measurements, Western blotting and partially denaturing SDS-PAGE.     

Isolation of a latent polyphenol oxidase from loquat fruit (Eriobotrya japonica Lindl.): kinetic characterization and comparison with the active form

Polyphenol oxidase (PPO) has been extracted from both soluble and particulate fractions of loquat fruit (Eriobotrya japonica Lindl. cv. Algerie). The soluble PPO (20% of total activity) was partially purified 3.3-fold after ammonium sulfate fractionation being in its active state. The particulate PPO fraction (80% of total activity) was purified to homogeneity in a latent form being activable by sodium dodecyl sulfate (SDS). The enzyme was purified 40.0-fold with a total yield of 15.3% after extraction by phase partitioning in Triton X-114 followed by three chromatographic steps. The molecular weight was estimated to be about 59.2 and 61.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography, respectively, indicating that latent PPO is a monomer. Latent PPO catalyzed the oxidation of chlorogenic acid (CA) at a rate 50-fold faster than that of 4-tert-butylcatechol (TBC) but the soluble active counterpart only twice. Both PPOs exhibited similar Km values for TBC but Km for CA was 5-fold higher for the latent than for the active soluble PPO. Other kinetic characteristics, including sensitivity to inhibitors, substrate specificity, thermal stability, temperature, and pH profiles, were quite different between both PPOs. These results provide strong evidences that the soluble active and the particulate latent are different forms of PPO in loquat fruit flesh. The results suggest that the major PPO form for the oxidation of CA, leading to enzymatic browning under physiological conditions, is the latent one.     

Hysteresis and positive cooperativity of iceberg lettuce polyphenol oxidase.

A kinetic study of the diphenolase activity of latent polyphenol oxidase (PPO), purified from Iceberg lettuce (Lactuca sativa L), revealed a sigmoid relationship between the reaction rate and the substrate concentration with a high Hill coefficient (n(H) = 3.8). This positive cooperativity had not been previously described for any PPO. Furthermore, the enzyme showed a lag phase in the expression of this activity, suggesting a hysteretic nature of the enzyme. The kinetic behavior, the latency and the lag phase varied at different steps of the purification process. PPO showed hyperbolic or cooperative kinetics depending on the pH assay and the sodium dodecyl sulfate (SDS) concentration. Substrate-induced slow conformational change of the oligomeric enzyme is suggested. The conformational change would be toward a more active enzyme form with higher affinity for the substrate and favoured by acid pH and SDS.     

Polyphenol oxidase overexpression in transgenic<Emphasis Type="Italic"> Populus</Emphasis> enhances resistance to herbivory by forest tent caterpillar (<Emphasis Type="Italic">Malacosoma disstria</Emphasis>)

In order to functionally analyze the predicted defensive role of leaf polyphenol oxidase (PPO; EC 1.10.3.1) in Populus, transgenic hybrid aspen (Populus tremula × P. alba) plants overexpressing a hybrid poplar (Populus trichocarpa × P. deltoides) PtdPPO1 gene were constructed. Regenerated transgenic plants showed high PPO enzyme activity, PtdPPO1 mRNA levels and PPO protein accumulation. In leaf disk bioassays, forest tent caterpillar (Malacosoma disstria) larvae feeding on PPO-overexpressing transgenics experienced significantly higher mortality and reduced average weight gain compared to larvae feeding on control leaves. However, this effect was observed only when older egg masses were used and the resulting larvae showed reduced growth and vigor. In choice tests, no effect of PPO overexpression was detected. Although PPO in poplar leaves is latent and requires activation with detergents or trypsin for full enzymatic activity, in caterpillar frass the enzyme was extracted in the fully activated form. This activation correlated with partial proteolytic cleavage, suggesting that PPO latency and activation during digestion could be an adaptive and defense-related feature of poplar PPO.     

Effect of detergents, trypsin and unsaturated fatty acids on latent loquat fruit polyphenol oxidase: basis for the enzyme's activity regulation

The effects of detergents, trypsin and fatty acids on structural and functional properties of a pure loquat fruit latent polyphenol oxidase have been studied in relation to its regulation. Anionic detergents activated PPO at pH 6.0 below critical micelle concentration (cmc), but inhibited at pH 4.5 well above cmc. This behavior is due to a detergent-induced pH profile alkaline shift, accompanied by changes of intrinsic fluorescence of the protein. Gel filtration experiments demonstrate the formation of PPO-SDS mixed micelles. Partial PPO proteolysis suggest that latent PPO losses an SDS micelle-interacting region but conserves an SDS monomer-interacting site. Unsaturated fatty acids inhibit PPO at pH 4.5, the strongest being linolenic acid while the weakest was gamma-linolenic acid for both, the native and the trypsin-treated PPO. Down-regulation of PPO activity by anionic amphiphiles is discussed based on both, the pH profile shift induced upon anionic amphiphile binding and the PPO interaction with negatively charged membranes.     

Characterization of polyphenol oxidase in coffee

Polyphenol oxidase (PPO) was characterized in partially purified extracts of leaves (PPO-L) and fruit endosperm (PPO-E) of coffee (Coffea arabica L.). PPO activity was higher in early developmental stages of both leaves and endosperm of fruits. Wounding or exposure of coffee leaves to methyl jasmonate increased PPO activity 1.5-4-fold. PPO was not latent and was not activated by protease treatment. PPO activity was stimulated 10-15% with sodium dodecyl sulphate (SDS) at 0.35-1.75 mM, but at higher concentrations activities were similar to the control samples, without detergent. Prolonged incubation of extracts with trypsin or proteinase K inhibited PPO activity but pepsin had no effect. Inhibition of PPO with proteinase K was increased in the presence of SDS. PPO activity from both tissues was optimal at pH 6-7 and at an assay temperature of 30 degrees C. Activity was highest with chlorogenic acid as substrate with a Km of 0.882 mM (PPO-L) and 2.27 mM (PPO-E). Hexadecyl trimethyl-ammonium bromide, polyvinylpyrrolidone 40. cinnamic acid and salicylhydroxamic acid inhibited PPO from both tissues. Both enzymes were inactivated by heat but the activity in endosperm extracts was more heat labile than that from leaves. The apparent Mr determined by gel filtration was 46 (PPO-L) and 50 kDa (PPO-E). Activity-stained SDS polyacrylamide gel electrophoresis (PAGE) gels and western blots probed with PPO antibodies suggested the existence of a 67 kDa PPO which is susceptible to proteolytic cleavage that generates a 45 kDa active form.     

Polyphenol oxidase: The chloroplast oxidase with no established function

Vaughn, K. C, Lax, A. R. and Duke, S. O. 1988. Polyphenol oxidase: The chloroplast oxidase with no established function. - Physiol. Plant. 72: 659–665.
Polyphenol oxidase (PPO) is an enzyme localized on the thylakoids of chloroplasts and in vesicles or other bodies in non-green plastid types. Although virtually all plastids contain PPO, little or no detectable activity is associated with guard cell and bundle sheath cell chloroplasts. Despite this nearly ubiquitous occurrence, no function for this enzyme has been established. The enzyme is nuclear-encoded and, unlike most chloroplast proteins is not encoded as a larger M, precursor molecule. This lack of a transit peptide sequence may be related to a unique mechanism of uptake, apparently involving inner envelope-derived vesicles. The M, range of most of the PPO forms is 36–45 kDa. PPO is apparently not involved in phenolic biosynthesis but is probably involved with the production of o -quinones during pathogen invasion. A role for PPO as an "oxygen buffer" is postulated, but little concrete data have been collected on any other functional role for this enzyme.     

低温与GA_3诱导菠萝黑心病的生理机制

菠萝黑心病是PPO催化氧化酚类物质形成褐色产物所致。低温或GA_3处理提高了PPO活性及其底物——儿茶酚、绿原酸和咖啡酸的含量,也导致了PAL活性增加;低温还使乙烯释放率增大。这些变化均有利于黑心病的发生和发展。     

Polyphenol oxidase and photosynthesis research

Very briefly, the present state of knowledge on the latent, lumen oriented polyphenol oxidase (PPO) of the chloroplast is reviewed. The location of PPO in the thylakoid membrane was described by D. Arnon 46 years ago. The N-terminus sequence of the spinach enzyme is reported. A historical sketch is given of the discovery of photophosphorylation and Arnon's visit to the admired O. Warburg.     

香蕉芒果果皮中酶活性与炭疽病菌潜伏侵染的关系

在树上正生长发育的香蕉果实,随着果实长大,果皮中过氧化物酶(POD)和多酚氧化酶(PPO)活性不断升高。采后的香蕉和芒果果实的果皮中,POD和PPO活性变化与呼吸代谢相关。苯丙氨酸解氨酶(PAL)主要分布在果皮中,并随着果实成熟度的提高,其活性呈极显著下降趋势。感病果实的果皮中三种酶活性均比健康果皮高。说明果皮中三种酶活性的变化与炭疽菌的潜伏侵染有关。     

Gel electrophoresis of polyphenol oxidase with instant identification by in situ blotting

Polyphenol oxidase (PPO) or tyrosinase is an important and ubiquitous enzyme responsible for browning in plants and melanization in animals. The molecular size of the plant PPO is varied among the species and its activity can be enhanced by a variety of anionic detergents. In the present study, we developed a simple method for the first-step identification of PPO in fruit and vegetable extracts. First, 3mm chromatographic paper was immersed in 0.5% (w/v) catechol solution as an immobilized PPO substrate. After running the extract with 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), one side of the glass plate was removed. The plate was immediately laid on top of the dried catechol-paper. A dark-brown band corresponding to PPO was visualized within 1 min and was further confirmed by a conventional Western blot using an antibody prepared against mushroom PPO. It also reveals that some vegetation (such as tomato, radish, and oriental melon) with low or no detectable activity in a conventional enzyme assay actually possessed marked levels of PPO activity when assessed by PAGE-blot. We propose that an inhibitor is associated with PPO in some plants; the inhibitor, however, is dissociated during the electrophoresis. Therefore, in addition to identify the molecular form of PPO, the present technique may explore the existence of PPO inhibitor(s) in plants. The detail of the method with respect to its relevance for searching a natural PPO inhibitor is described and discussed.     

PROPERTIES OF MEMBRANE-BOUND HEXOKINASE IN RAT BRAIN

Abstract— —-(1) The total hexokinase activity present in the mitochondrial fraction can be solubilized completely by incubation with salt and Triton X-100. This activity cannot be entirely released by washing with sucrose or by freezing and thawing.
(2) A part of the particle bound hexokinase exists in a latent form. The latent form is apparent after incubation with high salt concentrations, detergents or by freezing and thawing.
(3) Solubilization of membrane bound hexokinase is pH-dependent. Incubation in salt solutions increases the specific activity ten-fold. The salt concentration and pH are con-current. At pH 7.0 part of the hexokinase is solubilized. The lower the pH the less salt is required to release the same amount of activity.
(4) Triton X-100 solubilizes particle-bound hexokinase, but to a less extent than salts. The activation of hexokinase is greater with Triton X-100 than with salt.
(5) The possible nature of the bonds between hexokinase and mitochondrial membranes is discussed.     

烟草中多酚氧化酶(PPO)的特征

烟草中的多酚氧化酶介导的褐变会影响烟叶和烟丝的色泽和内在质量,因此对其特性的研究,以及活性的控制成为多年来的研究热点。本文从其生物发生模型、分子结构、生物化学和光谱学特征与植物抗病和机械损伤的关系,多酚氧化酶的抑制、多酚氧化酶的应用等方面着手,对近几年来烟草中PPO研究的最新成果进行总结和回顾,对一些有争议的问题进行了探讨,并对未来PPO研究的方向和领域进行了展望。     

Tentoxin stops the processing of polyphenol oxidase into an active protein

Previous studies in our laboratory have indicated that polyphenol oxidase (PPO), as measured by its activity, is not present in tentoxin-treated plants. In the present study, immunochemical techniques were used as a sensitive probe of tentoxin effects on PPO. Immunocytochemistry of PPO with peroxidase-antiperoxidase labelling techniques, revealed that in control Vicia faba L. chloroplasts, PPO was associated mainly with the thylakoids. Cytochemical staining of PPO activity using DL-dihydroxyphenylalanine (DOPA) as substrate was also localized only on the thylakoids in the control chloroplasts. In tentoxin-treated plants all of the immunologically-recognizable PPO accumulated at the plastid envelope although no PPO was detected by cytochemical methods. SDS polyacrylamide gels of extracts from control and tentoxin-treated Vicia leaves were blotted onto nitrocellulose and reacted with rabbit anti-PPO. Secondary labelling of the blots with goat-antirabbit IgG labelled with peroxidase revealed a 40 kdalton protein in both extracts. However, only the PPO from the control extract had DOPA oxidase activity. These data suggest that PPO accumulates in the plastid envelope membranes in tentoxin-affected cells and that PPO in these treated plants is not processed to an active protein.     

Location and catalytic characteristics of a multipotent bacterial polyphenol oxidase.

The melanogenic marine bacterium Marinomonas mediterranea contains a multipotent polyphenol oxidase (PPO) able to oxidize substrates characteristic for tyrosinase and laccase. Thus, this enzyme shows tyrosine hydroxylase activity and it catalyzes the oxidation of a wide variety of o-diphenol as well as o-methoxy-activated phenols. The study of its sensitivity to different inhibitors also revealed intermediate features between laccase and tyrosinase. It is similar to tyrosinases in its sensitivity to tropolone, but it resembles laccases in its resistance to cinnamic acid and phenylthiourea, and in its sensitivity to fluoride anion. This enzyme is mostly membrane-bound and can be solubilized either by non-ionic detergent or lipase treatments of the membrane. The expression of this enzymatic activity is growth-phase regulated, reaching a maximum in the stationary phase of bacterial growth, but L-tyrosine, Cu(II) ions, or 2,5-xylidine do not induce it. This enzyme can be separated from a second PPO form by gel permeation chromatography. The second PPO is located in the soluble fraction and shows a sodium dodecyl sulfate (SDS)-activated action on the characteristic substrates for tyrosinase, L-tyrosine, and L-dopa, but it does not show activity towards laccase-specific substrates. The involvement of the multipotent PPO in melanogenesis and its relationship with the SDS-activated form and with the alternative functions proposed for multicopper oxidases in other microorganisms are discussed.     

Involvement of UVB-induced reactive oxygen species in TGF-beta biosynthesis and activation in keratinocytes

TGF-beta produced by keratinocytes in response to UVB (290-320 nm) is a potential mediator for effects of acute and chronic solar radiation on skin. This study was designed to determine whether reactive oxygen species (ROS) mediate UVB-induced TGF-beta biosynthesis in keratinocytes and the subsequent activation of the latent TGF-beta complex. UVB irradiation elevated both total (latent plus active) and active TGF-beta in the keratinocyte supernatants, with a greater increase in the active form. UVB irradiation induced up to a 30% increase in ROS, and the ROS were detected up to 90 min after irradiation. NAC and Trolox, cytoplasmic ROS scavengers, abolished the UVB-induced TGF-beta and intracellular ROS, suggesting that UVB-induced ROS are involved in TGF-beta regulation. Inhibitors of NADPH oxidase activity, DPI and apocynin, decreased UVB-induced ROS. The increase in NADPH oxidase activity was mediated by EGFR activation. UVB-induced ROS also activated latent TGF-beta complex by stimulating MMP-2 and -9 activities. In summary, physiological doses of UVB increase intracellular ROS, which upregulate TGF-beta biosynthesis and activation of TGF-beta through increased activity of MMPs.     

A Dithiothreitol-Sensitive Tetrameric Protease from Spinach Thylakoids Has Polyphenol Oxidase Activity

Polyclonal antibody raised against a dithiothreitol-sen-sitivetetrameric protease (DSTP) from PSII membranes specificallyinhibited the polyphenol oxidase (PPO) activity of spinach thylakoids.DSTP was copurified with PPO activity on an affinity columnprepared with antibody against DSTP. These results suggest thatDSTP and PPO are the same protein. During purification of DSTP,Tween 20 was essential for stabilization of the protein, whichwas degraded in the absence of the detergent. Gel-filtrationchromatog-raphy of the purified DSTP revealed the presence of230-kDa (tetramer) and 60-kDa (monomer) species. The coppercontent of monomer species was determined to be 0.4 Cu atomper protein molecule, when the molecular weight of the proteinwas calculated to be 62,243, which is the value reported forspinach PPO [Hind et al. (1995) Biochemistry 34: 8157]. PurifiedDSTP caused the degradation as well as the dimerization of theextrinsic 23-kDa protein of PSII. The degradation of the proteinwas suppressed under anaerobic conditions induced by the presenceof glucose oxidase and glucose together. This fact suggeststhat oxygen molecules are involved in the proteolytic reactionand that the proteolytic activity and PPO activity may be correlatedwith each other. (Received September 27, 1996; Accepted December 4, 1996)     

Polyphenol oxidases in plants and fungi: going places? A review

The more recent reports on polyphenol oxidase in plants and fungi are reviewed. The main aspects considered are the structure, distribution, location and properties of polyphenol oxidase (PPO) as well as newly discovered inhibitors of the enzyme. Particular stress is given to the possible function of the enzyme. The cloning and characterization of a large number of PPOs is surveyed. Although the active site of the enzyme is conserved, the amino acid sequence shows very considerable variability among species. Most plants and fungi PPO have multiple forms of PPO. Expression of the genes coding for the enzyme is tissue specific and also developmentally controlled. Many inhibitors of PPO have been described, which belong to very diverse chemical structures; however, their usefulness for controlling PPO activity remains in doubt. The function of PPO still remains enigmatic. In plants the positive correlation between levels of PPO and the resistance to pathogens and herbivores is frequently observed, but convincing proof of a causal relationship, in most cases, still has not been published. Evidence for the induction of PPO in plants, particularly under conditions of stress and pathogen attack is considered, including the role of jasmonate in the induction process. A clear role of PPO in a least two biosynthetic processes has been clearly demonstrated. In both cases a very high degree of substrate specificity has been found. In fungi, the function of PPO is probably different from that in plants, but there is some evidence indicating that here too PPO has a role in defense against pathogens. PPO also may be a pathogenic factor during the attack of fungi on other organisms. Although many details about structure and probably function of PPO have been revealed in the period reviewed, some of the basic questions raised over the years remain to be answered.     

Isolation and Characterization of Strain MMB-1 (CECT 4803), a Novel Melanogenic Marine Bacterium

A novel marine melanogenic bacterium, strain MMB-1, was isolated from the Mediterranean Sea. The taxonomic characterization of this strain indicated that it belongs to the genus Alteromonas. Under in vivo conditions, L-tyrosine was the specific monophenolic precursor for melanin synthesis. This bacterium contained all types of activities associated with polyphenol oxidases (PPOs), cresolase (EC 1.18.14.1), catecholase (EC 1.10.3.1), and laccase (EC 1.10.3.2). These activities were due to the presence of two different PPOs. The first one showed all the enzymatic activities, but it was not involved in melanogenesis in vivo, since amelanogenic mutant strains obtained by nitrosoguanidine treatment contained levels of this PPO similar to that of the wild-type MMB-1 strain. The second PPO showed cresolase and catecholase activities but no laccase, and it was involved in melanogenesis, since this enzyme was lost in amelanogenic mutant strains. This PPO was strongly activated by sodium dodecyl sulfate below the critical micelle concentration, and it is a tyrosinase-like enzyme showing a lag period in its tyrosine hydroxylase activity that could be avoided by small amounts of L-dopa. This is the first report of a bacterium that contains two PPOs and also the first report of a pluripotent PPO showing all types of oxidase activities. The bacterium and the pluripotent PPO may be useful models for exploring the roles of PPOs in cellular physiology, aside from melanin formation. On the other hand, the high oxidizing capacity of the PPO for a wide range of substrates could make possible its application in phenolic biotransformations, food processing, or the cosmetic industry, where fungal and plant PPOs are being used.