Activation and alteration of plant and fungal polyphenoloxidase isoenzymes in sodium dodecylsulfate electrophoresis

Electrophoretic analysis of polyphenoloxidase isoenzymes from a variety of angiosperms and from mushroom revealed that the enzymes remain active in the presence of 0.1 % sodium dodecylsulfate. Electrophoresis in the presence of sodium dodecylsulfate allows the detection of latent enzyme forms of polyphenoloxidase, and can also convert slower migrating enzyme forms to faster migrating forms. Electrophoresis in the absence of sodium dodecylsulfate followed by incubation in the presence of sodium dodecylsulfate can also be used to detect latent forms of polyphenoloxidase. Together, these approaches provide a method for screening latent enzymes and give some insight into the mechanism of activation by sodium dodecylsulfate.     

Integrated antiherbivore, antidesiccant and ultraviolet screening properties of creosotebush resin

The surface leaf resin of creosotebush, Larrea spp. (Zygophyllaceae) exhibits antiherbivore, antidesiccant and UV screening properties. Composed mainly of monomeric phenolic aglycones, the resin absorbs most radiation of wavelength shorter than 410 nm, reduces the rate of evaporative water loss across cellulose membrane and exhibits tannin-like protein-complexing and digestibility-reducing properties. Leaf-chewing insects prefer mature leaves which have a lower resin content than young leaves. A phenoloxidaes system present in the leaves probably enhances the digestibility-reducing action of resin on in vitro proteolysis and growth-rates of Astroma quadrilobatum (Orthoptera: Proscopiidae) are negatively correlated with resin-phenoloxidase activity as measured by macerated leaf oxygen demand. Non-ideal behaviour of the resin is suggestive of cooperative action between components.     

Degradation of Cellular Phospholipids and Softening of Pressed Baker’s Yeast

Variations in lipid components of washings and homogenate of pressed baker’s yeast were investigated during the storage of pressed baker’s yeast at 30°C. Washings represents the substances which had leaked out from cells. Homogenate represents those contained in whole cells. Lipids in yeast washings increased toward softening, the phospholipids in yeast homogenate decreased continuously during storage. Two stages, an earlier period of storage (Stage I) and a later period of storage (Stage II) were observed in the degradation of phospholipids. Free fatty acid which was the main degradation product of phospholipid accumulated in Stage II, particularly at softening. The order in phospholipid degradation was PC>PE>PI + PS (PI>PS). Moreover, when washings of stored yeast at softening were assayed using ¹⁴C-acyl PC, the release of ¹⁴C-acyl fatty acid was observed.

These results suggest that phospholipids were degraded by some phospholipid-deacylating enzymes toward softening. From the results of lipid analysis, we inferred that the responsible enzymes were phospholipases.     

铜铁试剂对菜青虫多酚氧化酶的抑制作用

以菜青虫Pieris rapae (L.)为试材,冰浴匀浆,4℃下6 403 ×g 离心,取上清液,经35%饱和度(NH₄)₂SO₄沉淀,Sephadex G100凝胶过滤柱层析等分离步骤,获得部分纯化的菜青虫多酚氧化酶制剂。研究不同浓度铜离子及铜铁试剂对该酶的影响,结果表明：Cu₂₊在0～0.100 mmol/L范围内对酶活力表现激活作用;浓度大于0.125 mmol/L时表现抑制作用,其IC₅₀为0.651±0.022 mmol/L;铜铁试剂对该酶抑制作用的IC₅₀为0.100±0.012 mmol/L。抑制动力学研究结果表明：铜铁试剂对该酶表现为可逆抑制效应,为竞争性抑制类型,其抑制常数K_i为0.076±0.013 mmol/L。     

A critical evaluation of peroxidase profiles inParthenium argentatum

Specific localization of peroxidases after electrophoresis on nondenaturing polyacrylamide gels is discussed. The use of a multifunctional analysis for the separation of isoperoxidases from polyphenoloxidases is suggested.The Guayule Cooperative Programme of the Foundation for Research Development is thanked for its financial support.     

Purification and properties of polyphenoloxidase of mango peel (Mangifera indica)

Polyphenoloxidase from mango(Mangifera indica) peel was purified to homogeneity by ammonium sulphate fractionation, chromatography on DEAE-Sephadex and gel filtration of Sephadex G-200. The enzyme had an apparent molecular weight of 136,000. Its pH and temperature optimum were 5.4 and 50‡C, respectively. The enzyme possessed catecholase activity and was specific too-dihydroxy phenols. The enzyme also exhibited peroxidase activity. Some non-oxidizable phenolic compounds inhibited the enzyme competitively. High inhibitory effects were also shown by some metal chelators and reducing agents, Mango peel polyphenol oxidase when immobilized onto DEAE Sephadex showed slightly higher Km for catechol and lower pH and temperature optima.     

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.     

Polyphenol oxidase isoenzymes in avocado

Avocado polyphenol oxidase (PPO) was precipitated mainly in the 30–90% saturated ammonium sulfate fraction. The 40–75% saturated ammonium sulfate fraction (the partially purified enzyme) had the highest specific activity in the cultivars Lerman, Horeshim and Fuerte. The PPO was active towards o-dihydroxyphenols. Six active enzymes (a–f) were detected with D,L-DOPA, 4-methylcatechol, catechol, caffeic acid or chlorogenic acid. Band e was the most active in all cases. More isoenzyme bands (fast-moving) were observed with caffeic acid than with 4-methylcatechol. Furthermore, the isoenzyme patterns of the partially purified extracts of the cultivars could be distinguished with respect to caffeic acid.     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.