High PPO Activity Research Articles

An Indicating Role of Antioxidant System Enzymes at the Stage of Active Structural Anomalies Formation in Karelian Birch (Betula pendula Roth var. carelica (Mercl.) Hämet-Ahti).

A complex study of the antioxidant system enzymes (AOS) is an important subject of biochemical research; changes in the activity of these enzymes can be used as a biochemical marker of various processes in plants. At the same time, practically little attention has been paid to describing the regularities of these enzymatic reactions in different wood formation processes, such as xylogenesis. This article discusses the outcomes of different behaviors of AOS enzymes, which are involved in both the redistribution of the ROS balance and phenolic compounds at the early stages of wood formation in young plants of silver birch (Betula pendula Roth) with straight-grained wood and Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hamet-Ahti) with non-figured and figured parts within the single trunk. Spectrophotometric determination of AOS enzymes' activity can be used as a biochemical marker in the different wood formation processes, including xylogenesis. In this study, we studied structural anomalies of the woody plant trunk of Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hamet- Ahti). This study aimed to study AOS enzymes' activity in 12-year-old plants of silver birch (Betula pendula Roth) with straight-grained wood and Karelian birch (Betula pendula Roth var. carelica (Merckl.) Hamet-Ahti) with non-figured and figured parts within the single trunk. Plant tissues were ground in liquid nitrogen to a uniform mass and homogenized at 4°C in the buffer containing 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, 3 mM DTT, 5 mM MgCl2, and 0.5 mM PMSF. After 20 min extraction, the homogenate was centrifuged at 10000 g for 20 min (MPW-351R, Poland). The sediment was washed in the buffer thrice. The pooled supernatant and sediment were dialyzed at 4°C for 18-20 h against a tenfold diluted homogenization buffer. The enzymes' activity was determined spectrophotometrically (Spectrophotometer SF-2000, OKB Spectr, Russia). Proteins in the extracts were quantified by the method of Bradford. We observed different behaviors of the studied enzymes involved in both the redistribution of the ROS balance and phenolic compounds with subsequent lignification even at the early stages of wood formation in young plants and even in different trunk parts within a tree, which was consistent with results obtained earlier on adult plants. High SOD activity in the phloem compared to the activity in the xylem was accompanied by higher CAT activity. The POD/SOD ratio was significantly higher in the figured trunk parts in Karelian birch compared to other variants in the xylem and higher in Karelian birch plants compared to plants of common birch in the phloem. The CAT/POD ratio was significantly higher in plants with no signs of anomalies. The high POD and PPO activity in the xylem of figured trunk parts and in the phloem of figured and non-figured trunk parts of B. pendula var. carelica can be associated with the high activity of apoplast invertase. The study showed that at the stage of active formation of structural anomalies in the figured trunk parts in young plants of Karelian birch, hydrogen peroxide utilization occurred mainly due to increased POD activity. An increase in PPO activity in the trunk of figured plants could also be considered an indicator of the formation of structural anomalies. At the same time, in areas with developing abnormal wood, the POD/SOD ratio increased, and the CAT/POD ratio decreased, indicating a fine-tuning of the balance between superoxide radical and hydrogen peroxide, which, when changed, might regulate the rearrangement of xylogenesis towards proliferation in relation to differentiation.

Allergenicity of apple allergen Mal d 1 as effected by polyphenols and polyphenol oxidase due to enzymatic browning

Birch pollen-related apple allergy affects up to 5% of Germany's population due to Mal d 1. It has been hypothesized that apple allergenicity decreases by cutting, due to oxidation reactions of polyphenols with apple allergens. In this study, the influence of enzymatic browning on the allergenicity of apples was investigated by determining the total phenolic content, polyphenols, Mal d 1 and the activity of polyphenol oxidase in six different apple cultivars. Additionally, it was analysed how chlorogenic acid, caffeic acid and epicatechin combined with PPO affect rMal d 1 in vitro. Outcomes have shown that enzymatic browning led to a significant stronger decrease of Mal d 1 in Dülmener Rosenapfel, Ontario and Roter Boskoop compared to Braeburn, Golden Delicious and Granny Smith due to their stronger decrease in total polyphenols. High PPO activity may also reduce Mal d 1 allergenicity, even when the content of total polyphenols is low, as shown for Golden Delicious. The effect of PPO could be increased by naturally occurring contents of chlorogenic acid and caffeic acid as demonstrated in vitro. These results will be important for the breeding of apples with lower allergenicity.

Distribution and Characteristics of Polyphenoloxidase from Pacific White Shrimp (Litopenaeus vannamei).

Distribution of polyphenoloxidase (PPO) from different anatomical parts of Pacific white shrimp was examined. Among all parts, cephalothorax possessed the maximal PPO activity (P < 0.05), followed by pereopods, telson, pleopods, carapace, cuticle, and muscle, respectively. The higher PPO activity in cephalothorax was in line with the greater melanosis in this part during chilled storage. According to activity-staining toward 3,4-dihydroxy-ʟ-phenylalanine (ʟ-DOPA), PPO exhibited an activity band with a molecular weight (MW) of 210 kDa. When cephalothorax PPO was purified using ammonium sulfate precipitation and a series of chromatographic techniques, involving DEAE-Sepharose anion exchange and Sephadex G-75 gel filtration columns, homogeneity was obtained. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE, the Sephadex G-75 fraction showed a single band. The MW band on SDS-PAGE and gel filtration was estimated as 210 kDa, suggesting a monomeric molecule. For the inhibitor study, cysteine and 4-hexylresorcinol showed competitive inhibition toward PPO, while epigallocatechin gallate and kojic acid demonstrated mixed-type inhibition toward PPO. PRACTICAL APPLICATION: Melanosis (black spot formation) triggered by polyphenoloxidase (PPO) drastically reduces the shelf-life of shrimp. PPO was localized in several anatomical parts of Pacific white shrimp with varying activities. Certain compounds, including cysteine, 4-hexylresorcinol, epigallocatechin gallate, and kojic acid, showed PPO inhibitory activity with different modes of inhibition. The obtained information provided a promising method for manufacturers to keep the prime eating quality of Pacific white shrimp throughout postmortem transportation and storage using selected PPO inhibitors.

Effect of chitosan coatings with and without Stevia rebaudiana and modified atmosphere packaging on quality of cold stored fresh-cut apples

In this study, it was aimed to investigate the changes in polyphenoloxidase (PPO) activity, antioxidant properties and microbiological quality of fresh-cut apple slices coated with chitosan and stevia combinations under MAP (polypropylene-PP, 30 μm) storage at +1 °C for 3 days. The apples were cube-shaped and divided into three samples: i-Control (C, without coating), ii-Chitosan (CH, dipped into film mixture consisting of 0.75% chitosan, 1.5% glycerol and 2% ascorbic acid) and iii-Chitosan-Stevia combination (CHS, same CH film contains 2.5% stevia extract). PPO activity and microbiological (total yeast& mold-TYM and total psychrophilic aerobic bacteria-TPAB, total mesophilic aerobic bacteria-TMAB), total phenolic, antioxidant capacity, O2-CO2% and pH analyzes were performed. Results showed that PPO activity, O2-CO2% and pH values were lower, antioxidant substances were higher than C sample. At the end of the storage, CHS samples showed high PPO activity and antioxidant capacity values compared to CH. TMAB was found in C sample but it was under detectable limit (<2) during storage in CH and CHS samples; TPAB was found under detectable limit (<2) in all samples. Additionally, the chitosan and stevia have reduced the TYM counts. As conclusion; CH and CHS edible films are effective applications for reducing PPO activity and microbial load.

Identification of quantitative trait loci markers using diversity arrays technology for polyphenol oxidase activity in hexaploid wheat

ABSTRACTPolyphenol oxidase (PPO) causes undesirable darkening in wheat products such as Asian noodles. The inheritance of PPO activity was investigated in a doubled haploid (DH) population derived from a hybrid between a null PPO genotype VAW08-A17 and Australian wheat variety QALBis. The observed genetic ratio indicated three genes controlling high PPO activity. Quantitative trait loci (QTL) mapping was performed in this DH population using diversity arrays technology (DArT) to genotype the population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL with additive mode of action. Three closely associated DArT markers, wPt-7024, wPt-0094 and wPt-2544, were identified corresponding to these QTLs. Collectively, these loci explained 74% of the observed variation in PPO activity. Identification of DArT markers associated with PPO genes will accelerate the development of null or very low PPO cultivars that exhibit reduced time-dependent darkening of Asian noodles.

Growth, yield and physiology of mango (Mangifera indica L.) cultivars as affected by polyembryonic rootstocks

To assess the potential role of polyembryonic rootstocks on tree growth, yield and physiology of five mango cultivars, a study was conducted during 2013–15 on seven-year-old grafted trees. Rootstock K-5 inhibited canopy volume (CV) of Pusa Arunima (70.54%), Pusa Surya (27.64%) and Dushehari (30.15%) as of trees on Olour, while it was Olour which had inhibitory effect on CV of Amrapali (33.19%) and Mallika (64.11%) as of trees on K-5 rootstock. Kurakkan rootstock promoted highest yield in Amrapali and Pusa Surya, while both Kurakkan and Olour for Pusa Arunima; and K-5 and Kurakkan for Mallika seem to be more productive. The highest total chlorophyll content in Pusa Surya, Mallika and Dushehari was recorded on Olour rootstock, while both Kurakkan and Olour tended to show its higher values in Pusa Arunima, but Amrapali trees had the highest total chlorophyll content on Kurakkan. Photosynthetic rate (A) was promoted by Olour in Pusa Surya and Pusa Arunima, while K-5 rootstock induced higher A in Amrapali and Dushehari. Similarly, transpiration rate (E) was increased by Olour in Pusa Arunima, Pusa Surya and Amrapali. Moreover, Pusa Arunima and Amrapali had the highest activity of superoxide dismutase (SOD) on Kurakkan, while for Pusa Surya and Dushehari, it was highest on K-5 rootstock. Significantly higher peroxidase (POX) activity in Amrapali and Mallika was observed on K-5 rootstock, while glutathione recuctase (GR) activity was found higher in Amrapali and Mallika on Olour. Furthermore, both Olour and Kurakkan effectively upregulated polyphenoloxidase (PPO) activity in Pusa Arunima, though it was higher in Pusa Surya on K-5 and Kurakkan rootstocks. Trees of Amrapali had higher PPO activity on Kurakkan rootstock but Olour was found more effective for Mallika. Total phenolic compounds were higher in Pusa Arunima and Amrapali on Olour rootstock but it was K-5 which promoted their higher accumulation in Pusa Surya and Dushehari. Based on our findings it could be inferred that rootstocks alter most of the physio-chemical parameters of scion cultivars in mango though the extent of regulation of activities was scion specific.

The impact of catechin and epicatechin, total phenols and PPO activity on the Mal d 1 content in apple fruit

The most important apple allergen in Central Europe and North America is Mal d 1. Apples are a very important source of secondary plant metabolites like polyphenols in human nutrition. It is known that oxidised phenols can bind proteins. These irreversible bindings can lead to a reduced allergenicity. The most important phenols in apple are epicatechin, catechin and their polymeric structures, which have been identified as substrates of the polyphenoloxidase (PPO). The aim of this study was to analyse the influence of naturally occurring catechin and epicatechin contents in apple on the allergenicity of apple fruits. Fruits of the cultivars ‘Elstar’, ‘Diwa’ and ‘Boskoop’ were harvested and stored for 8 and 12weeks in a cold-chamber at 2°C. Mal d 1-, catechin-, epicatechin- and total phenol content as well as the activity of PPO were determined. Correlation analysis showed that naturally occurring catechin as well as epicatechin has no impact on the Mal d 1 content of the tested cultivars: correlation coefficient ranged from −0.203 to 0.501 for the correlation between Mal d 1 and catechin. The results further indicated that the activity of PPO is more important than the content of total phenols to reduce the Mal d 1 level. If there is a high PPO activity, Mal d 1 could be reduced even if the total phenol concentration is low.

Physico-chemical and antioxidant properties of four mango (Mangifera indica L.) cultivars in China

Four principal mango cultivars (Tainong No.1, Irwin, JinHwang and Keitt) grown in southern China were selected, and their physico-chemical and antioxidant properties were characterized and compared. Of all the four cultivars, Tainong No.1 had highest content of total phenols, ρ-coumaric acid, sinapic acid, quercetin, titratable acidity, citric acid, malic acid, fructose, higher antioxidant activities (DPPH, FRAP) and L∗, lower pH, PPO activity and individual weight. Keitt mangoes showed significantly (p<0.05) higher contents of β-carotene, ρ-hydroxybenzoic acid, sucrose, total sugar, total soluble solid, catechin, succinic acid and higher PPO activity. JinHwang mangoes exhibited significantly (p<0.05) higher individual weight and PPO activity, but had lower content of total phenols, β-carotene and lower antioxidant activity. Principal component analysis (PCA) allowed the four mango cultivars to be differentiated clearly based on all these physico-chemical and antioxidant properties determined in the study.

Effect of solution pH of cysteine-based pre-treatments to prevent browning of fresh-cut artichokes

Fresh-cut artichokes were treated with cysteine in the form of aqueous solutions of l-cysteine hydrochloride monohydrate at 0.5% (w/v) normal pH 2.2 adjusted to different pH values with NaOH (from 2 to 7). General appearance and colour measurements during storage at 5°C showed different degrees of suppression of darkening on cut surfaces. Cysteine pre-treatments caused an increase in the yellow index (b* value) of fresh-cut artichokes compared to untreated samples, which was more pronounced at lower pH (47.9 versus the initial value of 28.3 after 1 day). Artichokes treated at lower pH values also had a lower appearance score and higher PPO activity than artichokes treated at higher values, closer to neutrality. Starting from 3 days of storage a negative correlation between solution pH and PPO activity was also observed, that for artichokes treated with cysteine at pH 7 was lower than for control samples. l-cysteine pre-treatments did not affect total phenolic content and antioxidant activity of fresh-cut artichokes, which showed an increasing pattern after 8 days. These data may represent an important step in prevention of browning in fresh-cut artichokes.

Molecular and physico-chemical evaluation of enzymatic browning of whole meal and dough in a collection of tetraploid wheats

Many studies have demonstrated the role of polyphenol oxidase (PPO; E.C. 1.14.18.1) in darkening of wheat products. Even slight browning of pasta is a major hindrance to consumer acceptance. The aim of this research was to evaluate the variability of PPO activity, browning level, and protein content in a collection of more than 100 genotypes of tetraploid wheat, including cultivars and landraces. A molecular approach was followed to evaluate the efficiency of marker PPO18, discovered and used in common wheat, in detecting tetraploid wheat genotypes with low browning level. The data showed a significant genotype influence on the activity of the PPO enzyme, which was correlated with brown index of whole meal and dough, and with protein content. On the whole, the cultivars showed lower PPO activity, brown index, and protein content than landraces, but wide variability was present. Marker PPO18 detected four different alleles for Ppo-A1. The allele Ppo-A1f identified genotypes with high PPO activity and brown index, while the Ppo-A1b and null alleles were associated with the opposite characteristics. Hence, this marker can be used to select new tetraploid wheat cultivars with low browning level.

Rooting behaviour, polyphenol oxidase activity, and biochemical changes in grape rootstocks at different growth stages

Four grape rootstocks belonging to different Vitis species were planted in September. Rooting behaviour, polyphenol oxidase (PPO; EC 1.14.18.1) activity and biochemical parameters were studied at different growth stages after planting the cuttings in polythene bags. Significant differences were recorded for rooting success among the different rootstocks, with the maximum sprouting percentage determined in the Freedom rootstock. The highest variation in PPO activity was also recorded in Freedom. High PPO activity was recorded in rootstock 140 Ru, while the highest root length was recorded in Dog Ridge. In the rootstock 110 R, the PPO activity was the lowest during the initial stage, though it increased up to 60 days after planting (DAP) and was then reduced up to 90 DAP. Increases in the number of rooting primordials were recorded in all the different rootstocks at different stages of growth. A higher number of rooting primordials was recorded in Freedom, and it was followed by 110 R. The present study suggests the differences in the rooting behaviour of the different rootstocks are based on PPO activity at regular time intervals up to 90 DAP.

Screening quality and browning susceptibility of five artichoke cultivars for fresh‐cut processing

AbstractBACKGROUND: Artichoke is a rich source of bioactive compounds, mainly phenols, in the Mediterranean diet, but its consumption is limited by the complexity of time‐consuming trimming operations. Fresh‐cut processing would therefore add convenience to its consumption, even though the severity of post‐cutting browning of artichoke pieces is still a major problem. Since susceptibility to browning may vary widely among genotypes, the choice of the cultivar is a very critical step in the fresh‐cut process. In this study, five different Italian cultivars (C3, Catanese, Tema, Violetto Foggiano and Violetto Sardo) were screened for their initial quality and composition, and their post‐cutting performance during storage at 5 °C and 20 °C.RESULTS: C3 showed the highest phenol content (3.4 g GA kg−1) and antioxidant activity (24.5 mmol L−1 kg−1), but the worst quality in terms of appearance and colour changes, also due to its high PPO activity (62.2 U g−1). Catanese showed the highest vitamin C content (117.7 mg kg−1), the lowest phenol content (1.8 g GA kg−1), and the best post‐cutting quality. Tema, Violetto Foggiano and Violetto Sardo showed an intermediate phenol content, the latter showing the lowest appearance score after C3.CONCLUSION: These results confirmed the role of phenols in browning processes of fresh‐cut artichokes, giving the first available information on artichoke cultivar suitability to be processed as a fresh‐cut product. Copyright © 2009 Society of Chemical Industry

Classification of wheat PPO genes and effect of non-synonymous cSNP on kernel PPO activity

AbstractPolyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products. In this study, wheat PPO sequences (mRNA) were searched/BLASTed in the NCBI database and aligned using DNAMAN software. The results showed that wheat PPO genes could be divided into two clusters (I and II) and that three genes (‘i’) of cluster II seemed not to be located on chromosomes 2A and 2D. Ninety-four single nucleotide polymorphisms (SNPs) were detected between two haplotypes of the PPO gene on chromosome 2D. Eighty of these were found in the coding region (coding (c) SNPs) and 36 were non-synonymous cSNPs, which could affect the PPO amino acid sequence. Primers (STS-H) were designed at some non-synonymous cSNPs sites and were used to investigate the correlations between allelic variants and PPO activity of seeds – a total of 130 common wheat varieties were evaluated in 2 years. The results showed that STS-H could amplify a 460 bp DNA fragment in most cultivars with high PPO activity, while no PCR product was detected in most cultivars with low PPO activity. To improve the selection efficiency of a single dominance molecular marker, the multiplex polymerase chain reaction (PCR) system of STS-H and STS01 markers was also studied, based on the complementary between them.

Polyphenoloxidase is induced by methyljasmonate and Meloidogyne javanica in soybean roots but is not involved in resistance

Abstract The role of the enzyme polyphenoloxidase (PPO) in the response of two soybean varieties, Hartwig (resistant) and Cristalina (susceptible), to Meloidogyne javanica was studied in plants where root systems were exposed to the known PPO inducer, methyljasmonate. Chlorogenic acid was the best substrate for root PPO. Treatment of both varieties with 100 and 400 μM methyljasmonate solutions induced a similar increase in enzyme activity 72 h after treatment. Inoculation of roots with second-stage juveniles (J2) induced PPO increase in cv. Cristalina but not in cv. Hartwig. Moreover, combined treatment of methyljasmonate and J2 inoculation enhanced PPO activity in both varieties. Two PPO cDNAs were isolated from the roots of the resistant variety 48 h after J2 inoculation, and Southern blot experiments in both varieties, using GmPPOJH1 and GmPPOJH2 cDNAs as probes, indicated that PPOs are represented by a multigene family in soybean. RT-PCR assays showed more GmPPOJH1 transcripts in plants treated with methyljasmonate and infected with nematodes, where high PPO activity was also observed. Plants treated with methyljasmonate and infected with J2 showed a marked decrease of nematode population 35 days after inoculation. These findings suggest that methyljasmonate triggers a resistant response in soybean roots to M. javanica but PPO is not involved in the resistance process.

Systemic Effects on Oxidative Enzymes inPhaseolus vulgarisLeaves That Have Been Wounded by the GrasshopperMelanoplus differentialis(Thomas) or Have Had a Foliar Application of Jasmonic Acid

Fifteen‐day‐old intact primary leaves of Phaseolus vulgaris were either treated with 1 mM (−)−jasmonic acid (JA) or were wounded by fourth instar grasshoppers. Changes in protein content and the oxidative enzymatic activity of lipoxygenase‐3 (lox‐3), polyphenol oxidase (ppo), and peroxidase (pod) in both the trifoliate and primary leaves of the wounded plant were measured at 0, 24, 48, and 72 h using spectrophotometric methods. Results indicated that both JA treatment (at a higher rate) and wounding significantly enhanced total protein content in primary leaves at 24 and 48 h and induced systemic protein synthesis in trifoliate leaves up to 48 h. Insect wounding in primary leaves generally induced significant ppo, pod, and lox‐3 activity throughout the 72 h, peaking at levels of 227% at 48 h, 56% at 72 h, and 37% at 24 h, respectively, compared to the control. In the trifoliate leaves of insect‐wounded primary leaves, high systemic lox, ppo, and pod activity was sustained throughout the 72 h. In primary leaves treated with JA, lox and ppo generally maintained high activity up to 48 h. The pod activity, however, was suppressed throughout the 72 h. In the trifoliate leaves of JA‐treated primary leaves, high lox activity was consistently retained throughout the 72 h, high ppo activity was maintained up to 48 h, and high pod activity was observed up to 24 h. In general, the effects of JA treatment and grasshopper wounding on enhanced protein synthesis and enzyme activity at the point of injury and on trifoliate leaves indicate that when wounded, signals are systemically mobilized.

Some factors affecting Norway lobster (Nephrops norvegicus) cuticle polyphenol oxidase activity and blackspot development

SummaryPolyphenol oxidase (PPO) activity was measured in the cuticles of Norway lobsters caught over a period of 7 months. Increased PPO activity was observed between early August and early October 1995, coinciding with one of the reported annual periods of peak moulting activity, although a high PPO activity detected in May may have marked the spring moulting period. A relationship was found between the number of claws remaining on the Norway lobsters and the PPO activity, suggesting that exposing Norway lobsters to damage and stress is associated with an increase in PPO activity. No correlation was observed between initial PPO activity and blackspot development during storage of the Norway lobsters. This indicates that biochemical events occurring during storage are more important to blackspot development than the initial PPO level.

Leaf effects of wild species ofSolanum and interspecific hybrids on growth and behaviour of the potato tuber moth,Phthorimaea operculella Zeller

Four introductions ofSolanum berthaultii wild species and sevenS. tuberosumxS. berthaultii hybrids were evaluated by antibiosis and antixenosis laboratory tests with potato tuber moth. Some morphological and chemical traits related to the trichomes were analysed. Two clones of the wild speciesS. sparsipilum and five ofS. pinnatisectum, characterised by high tuber resistance, were tested for leaf resistance, both by antibiosis and antixenosis assays. The genotypes with high density of trichomes A and with high exudate PPO activity, showed a moderate negative effect on pupal weight and fecundity in the antibiosis tests. Trichomes B exudate showed a strong repulsive effect on oviposition of adult in the antixenosis tests. TheS. pinnatisectum clones revealed a good leaf resistance level, whereas theS. sparsipilum clones showed lower levels of antibiosis and antixenosis.

Cloning of a Human cDNA for Protoporphyrinogen Oxidase by Complementation in Vivo of a hemG Mutant of Escherichia coli

Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that catalyzes in the penultimate step in the heme biosynthetic pathway. Hemes are essential components of redox enzymes, such as cytochromes. Thus, a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration and grows poorly even in rich medium. By complementation with a human placental cDNA library, we were able to isolate a clone that enhanced the poor growth of such a hemG mutant strain. The clone encoded the gene for human PPO. Sequence analysis revealed that PPO consists of 477 amino acids with a calculated molecular mass of 50.8 kilodaltons. The deduced protein exhibited a high degree of homology over its entire length to the amino acid sequence of PPO encoded by the hemY gene of Bacillus subtilis. The NH2-terminal amino acid sequence of the deduced PPO contains a conserved amino acid sequence that forms the dinucleotide-binding site in many flavin-containing proteins. Northern blot analysis revealed the synthesis of a 1.8-kilobase pair mRNA for PPO. A homogenate of the monkey kidney COS-1 cells that had been transfected with the cDNA had much higher PPO activity than an extract of control cells, and this activity was inhibited by acifluorfen, a specific inhibitor of PPO. Furthermore, the cDNA was expressed in vitro as 51-kilodalton protein, and after incubation with isolated mitochondria the protein was found to be located in the mitochondria, having just the same size as before, an indication that PPO is a mitochondrial enzyme and has no apparent transport-specific leader sequence.

Polyphenoloxidase-activity and -activation in embryogenic and non-embryogenic suspension cultures of Euphorbia pulcherrima

The activity and activation potential of polyphenoloxidase (PPO, E.C. 1.10.3.1.) of tissue from shoot tips, adult leaves and embryogenic and non-embryogenic cell suspension cultures of Euphorbia pulcherrima was investigated using an oxygen probe technique. PPO derived from differentiated in vivo plant tissue (shoot tips, leaves) cannot be activated either by storage at 0-4°, freezing and thawing, incubation with CaCl2, sodium dodecyl sulfate or by incubation with trypsin. Embryogenic cells are characterized by high initial PPO activity and strong activation potential of membrane bound enzyme. Non-embryogenic material reveals low phenolase activity and low activation potential. An activation quotient (based on the ratio between "PPO-activity determined after sodium dodecyl sulfate incubation" to "PPO-activity determined after CaCl2-incubation") was calculated. This is independent of absolute enzyme activity and can be used for characterization of the embryogenic status of cells.

CHARACTERIZATION AND INHIBITION OF POLYPHENOL OXIDASE FROM PEARS (PYRUS COMMUNIS L. CV. BOSC AND RED)

Red pears had higher PPO activity, total phenolics and chlorogenic acid concentration than Bosc pears. PPO activity, and phenolics both decreased in fruits held at room temperature. pH and temperature optima for Bosc and Red pears PPOs were 5.0 and 5.5, and 20 and 23 C, respectively. 4-Methylcatechol, catechol and dopamine were good substrates for PPO from both pear cultivars; however, no activity was observed with any of the mono-hydroxy substrates studied. Higher Km and lower Vmax values were observed for Bosc pear PPO. Heating at 75 C for 30 min completely inactivated the enzyme from both cultivars. Heating at 55 and 65 C for the same duration resulted in partial inactivation (45-60%) of this enzyme. Ascorbic acid, L-cysteine, sodium metabisulfite and thiourea effectively inhibited browning due to pear PPOs.