Some kinetic properties of polyphenol oxidase from Thymbra spicata L. var. spicata

Polyphenol oxidase (PPO) of Thymbra (Thymbra spicata L. var. spicata) was isolated by (NH₄)₂SO₄ precipitation and dialysis. A diphenolase from Thymbra plant, active against 4-methylcatechol, catechol and pyrogallol was characterized in detail in terms of pH and temperature optima, stability, kinetic parameters and inhibition behaviour towards some general PPO inhibitors. 4-Methylcatechol was the most suitable substrate, due to the lowest K_m and the biggest V_max/K_m values, followed by catechol and pyrogallol. The Thymbra PPO had maximum activity at pH 5.0, 7.0 and 8.0 with 4-methylcatechol, catechol and pyrogallol substrates, respectively. The optimum temperature of activity for Thymbra PPO was 30, 40 and 50 °C for 4-methylcatechol, catechol and pyrogallol substrates, respectively. It was found that optimum temperature and pH were substrate-dependent studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time. Inhibition of Thymbra PPO was investigated with inhibitors such as l-cysteine and glutathione using 4-methylcatechol, catechol and pyrogallol as substrates. It was found that l-cysteine was a more effective inhibitor than glutathione owing to lower K_i. The type of inhibition depended on the origin of the PPO studied and also on the substrate used. Furthermore, the IC₅₀ values of inhibitors sudied on PPO were determined by means of activity percentage (I) diagrams.     

Epinephrine quantification in pharmaceutical formulations utilizing plant tissue biosensors

A plant tissue biosensor associated with flow injection analysis is proposed to determine epinephrine in pharmaceutical samples. The polyphenol oxidase enzymes present in the fibers of a palm tree fruits (Livistona chinensis), catalyses the oxidation of epinephrine to epinephrinequinone as a primary product. This product is then electrochemically reduced (at −0.10 V versus Ag/AgCl_sat) on the biosensor surface and the resulting current is used for the quantification of epinephrine. The biosensor provides a linear response for epinephrine in the concentration range from 5.0 × 10⁻⁵ to 3.5 × 10⁻⁴ mol l⁻¹. The limit of detection estimated for this interval was 1.5 × 10⁻⁵ mol l⁻¹ and the correlation coefficient of 0.998, working under a flow rate of 2.0 ml min⁻¹ and using a sample loop of 100 μl. The repeatability (R.S.D. for 10 consecutive determinations of a 3.0 × 10⁻⁴ mol l⁻¹ epinephrine solution) was 3.1%. The results obtained by the method here proposed were compared with the official UV spectrophotometric procedure and also using a plant tissue reactor. The responses obtained with the proposed strategies were in good agreement with both ways of analyses, whereas the values obtained by the official spectrophotometric method was strongly affected by benzoic acid, present in the formulation of pharmaceutical product utilized for inhalation. Such favorable results obtained with the carbon paste biosensor or utilizing the bioreactor, joined with the simplicity of its preparation turns these procedures very attractive for epinephrine quantification in pharmaceutical products.     

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30°C respectively and K_m and V_max values were 7.90?×?10^?4?M, and 11290?EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, β-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a K_i value of 1.79?×?10^?9?M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.     

Purification of a polyphenol oxidase isoform from potato (Solanum tuberosum) tubers

A different expression pattern of polyphenol oxidases has been observed during storage in cultivars of potato (Solanum tuberosum L.) featuring different length of dormancy: a short-dormant cultivar showed, at the end of the dormancy, both the highest polyphenol oxidase activity and the largest number of enzyme isoforms. An isoform of polyphenol oxidase isolated at the end of the physiological dormancy from a short-dormant cultivar has been purified to homogeneity by means of column chromatography on phenyl Sepharose and on Superdex 200. The purification factor has been determined equal to 88, and the molecular mass of the purified isoform has been estimated to be 69 and 340 kDa by SDS polyacrylamide gel electrophoresis and gel filtration on Superdex 200, respectively, indicating this PPO isoform as a multimer. The corresponding zymogram features a diffused single band at the cathodic region of the gel and the pI of this polyphenol oxidase has been calculated equal to 6.5.     

The use of diversity profiling to characterize chemical modulators of the histone deacetylases

Target specificity and off-target liabilities are routinely monitored during the early phases of drug discovery for most kinase projects. Typically these criteria are evaluated using a profiling panel comprised of a diverse collection of in vitro kinase assays and relates compound structure to potency and selectivity. The success of these efforts has led to the design of similar panels for phosphatase, protease, and epigenetic targets. Here the implementation of an epigenetic profiling panel, comprised of eleven histone deacetylases (HDACs) and one histone acetyltransferase (HAT), was used to evaluate chemical modulators of these enzymes. HDAC inhibitors (HDACi) such as sodium butyrate and trichostatin A demonstrate diverse biological effects which have led to broad speculation about their therapeutic potential in multiple disease states. Some HDACi have demonstrated tumor suppression in vivo and recently Zolinza was the first HDACi approved by the FDA for the treatment of cutaneous T-cell lymphoma. While HDACi have demonstrated therapeutic utility, many of the first generation compounds are pan-inhibitors. Thus, use of an HDAC profiling panel will be essential in achieving isoform specificity of the next generation of inhibitors. To this end, twenty-one compounds, twelve of which are known to have activities against the HDACs, were tested to evaluate the utility of the epigenetic panel. Additionally, these compounds were tested against a larger 72 member enzyme panel comprised of kinase, phosphatase and protease activities. This effort represents the first time these compounds have been profiled with such a broad range of biochemical activities.     

采后荔枝果皮色素、总酚及有关酶活性的变化

成熟度约八成的“淮枝”在采收后2天内,色素的合成代谢仍相当活跃,类胡萝卜素,花色素苷的含量增高,多酚氧化酶和苯丙氨酸解氨酶的活性增大,总酚保持原有水平,叶绿素降解,反映了果实达到完全成熟的特征,随后,上述各项参数因果实的衰老而下降,与采收当天相比,7天后花色素苷为90％,类黄酮为59％,总酚为71％,苯丙氨酸解氨酶活性为46％,讨论了酚类,花色素苷及两种酶活性之间的关系及对荔枝果实在成熟,衰老褐变中的作用。     

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.     

大孔树脂纯化嘉宝果叶片多酚及其生物活性和组成分析

为探讨嘉宝果(Myrciaria cauliflora)叶片多酚的分离纯化方法,对4种树脂(NKA-2、NKA-9、HPD-826和HPD-400A)进行了筛选,并分析了其多酚的抗氧化、体外降糖活性和组成成分。结果表明,NKA-9树脂适于嘉宝果叶片多酚纯化,最佳工艺条件为:上样液质量浓度2.00 mg/mL、洗脱液乙醇体积分数70%、上样流速1.0 mL/min、上样量204 mL、洗脱流速0.9 mL/min、洗脱量70 mL。嘉宝果叶多酚纯度可达69.86%。嘉宝果叶片纯化后的多酚抗氧化及α-葡萄糖苷酶抑制活性高于纯化前,但α-淀粉酶抑制活性低于纯化前。HPLC结果表明,嘉宝果叶片中含有杨梅苷、芦丁、金丝桃苷和鞣花酸,其中鞣花酸含量最高[(16.15±0.49) mg/g]。因此,NKA-9树脂适合分离纯化嘉宝果叶片多酚,纯化后的多酚抗氧化及α-葡萄糖苷酶抑制活性增强。     

间苯二酚、水杨酸对绿豆下胚轴不定根形成的作用

２０—１００ｍｇＬ（－１）间苯二酚能明显地促进绿豆下胚轴不定根的形成,与２０ｍｇＬ（－１）ＩＢＡ混合处理具加成效应,其作用在于降低生根初期ＩＡＡ氧化酶和多酚氧化酶活性．１０—１００ｍｇＬ（－１）水杨酸抑制下胚轴不定根的形成,随处理浓度的加大,对生根数目、生根范围和根重的抑制作用增加．水杨酸处理后１－３ｄ,能提高ＩＡＡ氧化酶和多酚氧化酶的活性．     

Import of polyphenol oxidase by chloroplasts is enhanced by methyl jasmonate

Polyphenol oxidase (PPO; EC 1.10.3.2 or EC 1.14.18.1) takes part in the response of tomato plants (Lycopersicon esculentum Mill.) to wounding and herbivore attack, mediated by the octadecanoid wound-signaling pathway. Wounding and methyl jasmonate (MeJA) induce expression of ppo genes and markedly increase the level of the enzyme. We report that pretreatment with MeJA also markedly increased the ability of isolated tomato chloroplasts to import and process PPO precursors (pPPO). Pea (Pisum sativum L.) chloroplasts showed no such response. Wounding or ethylene alone was ineffective but ethylene was synergistic with MeJA. Treatment with MeJA conferred a strong binding of pPPO, or its processing intermediate, to thylakoids and subsequent translocation into the lumen and processing to the mature protein. The effect on PPO import and translocation was evident after 8–16 h exposure to MeJA. Membrane-bound pPPO was cross-linked to a proteinaceous component of the thylakoid translocation apparatus, apparently induced by MeJA. The import and processing of other nuclear-encoded thylakoid proteins were not affected by MeJA in tomato. A 90-kDa protein that co-fractionated with thylakoids was induced along with the increase in competence for PPO import, and was identified as the proteinase-inhibitor multicystatin. It is concluded that the 90-kDa protein observed is part of the MeJA-induced defense response of tomato, not a component of the thylakoid translocation apparatus.Abbreviations Chl Chlorophyll - i and p Prefixes used to denote the intermediate and precursor forms of a protein, respectively - JA Jasmonic acid - LSU Large subunit of Rubisco - MeJA Methyl jasmonate - OE23 and OE33 23- and 33-kDa subunits of the oxygen-evolving complex of PSII - PC Plastocyanin - pPPO (iPPO, PPO) Precursor (intermediate, mature) form of polyphenol oxidase