O-dihydric Phenols Research Articles

Comparative Biochemical Analysis of Secondary Metabolites in Commonly Grown Legume Varieties

In this research study, quantitative determination of phytoconstituents in seed coat, dehusked raw dal, cooked dal and whole seed of locally grown pulses viz. pigeonpea, lentil, desi and kabuli chickpea, and ricebean was carried out. Among the four treatments of all the pulses, highest yield in methanol was found in ricebean. Total phenolic content (mg GAEg-1) varied from 1.72-103.62 in all of the extracts. Similarly, flavonoids (mg CAEg-1) ranged from 1.13 to 33.66 which also found to be reduced in dehulled and cooked extracts. o-Dihydric phenols, hydrophobic and hydrophilic phenols followed order as: seed coat> whole seed> raw dal> cooked dal. Results obtained by DPPH method showed that free radical scavenging capacities ranged from 53.80 to 92.00% while antioxidant activity ranged from 51.00 to 78.60% by FTC method. A highly significant correlation (plessthan0.05) between antioxidant activities/free radical scavenging efficiencies and phenolic contents as well as flavonoids was observed in most of the extracts under study. The results concluded that the pulse seed extracts may be valuable natural source of secondary metabolites for nutraceutical industry.

Morphological and antioxidant characteristics of mulberry (Morus spp.) genotypes

Abstract Mulberry is a multipurpose tree, which can be utilized for catering diversified needs such as food, fodder, fuel and fibre. It has earned ‘superfood’ status, in European countries, owing to presence of bioactive compounds in fruits. In India, mulberry is considered an underutilized fruit crop and the breeding efforts on mulberries made so far were restricted to development of genotypes suitable for sericulture. However promising selections of mulberry, intended for commercial fruit production, have recently been made. Therefore, an investigation about the phytochemical constituents of available promising mulberry genotypes could help develop an improved understanding and appreciation of the nutraceutical and medicinal value of this fruit, which consequently will promote its consumption in society. In the present investigation, we assessed variations in morphological characteristics, phytochemical and antioxidant capacity among 10 selected mulberry genotypes belonging to 3 mulberry species (Morus alba, M. rubra and M. laevigata). The antioxidant capacity was estimated by CUPRAC, FRAP and DPPH assays. The ascorbic acid content varied from 6.8 to 27.1 mg 100 g−1, total polyphenols from 0.51 to 1.58 mg g−1, total flavonoids from 0.37 to 1.26 mg g−1 and O-dihydric phenol from 0.14 to 0.33 mg g−1 fresh weight. The average antioxidant activities were 3.02–6.92 and 2.79–5.70 µM TE g−1 as determined by the CUPRAC and FRAP assays, respectively. Likewise, according to the results obtained, genotype Delhi Local was found to exhibit the strongest DPPH free-radical scavenging activity followed by Thar Lohit. The results of the study showed that the dark red-fruited mulberry ‘Delhi Local’ with higher antioxidant potential appears to be promising for further commercial exploitation.

Phytochemical Constituents and Antioxidant Activities of Some Indian Jujube (Ziziphus mauritiana Lamk.) Cultivars

Twenty-eight varieties of Indian jujube were estimated for various health-promoting compounds such as ascorbic acid, total flavonoids, flavanol, O-dihydric phenol and total phenolics. The antioxidant capacity was also measured by cupric reducing antioxidant capacity (CUPRAC), ferric reducing antioxidant power (FRAP) and 1,1 diphenyl-2-picryl hydrazyl (DPPH) assays. The ascorbic acid content varied from 47.81 to 160.12 mg/100 g, total phenolics from 48.69 to 196.34 mg/100 g, total flavonoids from 60.32 to 173.11 mg/100 g, flavanol from 25.21 to 70.59 mg/100 g and O-dihydric phenol from 5.03 to 19.26 mg/100 g fresh weight. The average antioxidant activities were 1.6–6.33 and 1.22–5.49 μM TE/g as determined by the CUPRAC and FRAP assays, respectively. Likewise, according to the results obtained, cv. ZG-3 was found to exhibit the strongest DPPH free radical scavenging activity followed by Katha Phal and Thar Sevika. The study also revealed a considerable amount of variation among the genotypes tested in relation to their phenolic content and antioxidant activity. Practical Applications Numerous studies have shown that fruits are a rich source of diverse nutrients and non-nutrient molecules, many of which display antioxidant properties. Indian jujube or ber is an important fruit crop of the hot arid regions in India as it forms an integral part of the life of the locals as a source of nutrition and other purposes. It is therefore important to assess different Indian jujube varieties for the content of various antioxidants in order to better identify their overall nutritional value and to encourage its increased consumption by the general public.

Biomolecules involved in resistance againstChilo partellus(Swinhoe) inSorghum bicolorL.

The phenolic profile and specific activity of oxidative enzymes in sorghum leaves and stem resistant and susceptible to Chilo partellus (Swinhoe) were analyzed at 45 and 60 days after germination. Resistant varieties had higher content of total phenols, o-dihydric phenols, flavanols and tannins than the susceptible varieties at both the stages of plant growth and the concentration of these biomolecules increased after infestation as well as with plant age in both sets of varieties. Moreover, resistant varieties exhibited higher specific activity of polyphenol oxidase, peroxidase, phenylalanine ammonia-lyase and tyrosine ammonia-lyase in their leaves and stem in comparison to the corresponding activities in susceptible ones. Peroxidase activity was several folds than the polyphenol oxidase. Healthy leaves of susceptible varieties showed higher catalase activity in comparison with resistant ones at day 45 and this trend was reversed at day 60. Role of phenolic compounds and oxidative enzymes particularly the peroxidase in determining resistance against stem borer has been high lighted.

The Effects of Nitric Oxide on the Oxidations ofl-Dopa and Dopamine Mediated by Tyrosinase and Peroxidase

The effects of nitric oxide (NO) on both tyrosinase/O(2)- and horseradish peroxidase/H(2)O(2)-mediated oxidations of dopamine and its o-dihydric phenol precursor l-dopa were compared with autoxidative processes and quantitatively assessed by oxidative and reductive electrochemical detection systems. In peroxidase/H(2)O(2)/NO-catalyzed reactions, significantly more substrate was oxidized than in the corresponding control incubations lacking NO. In tyrosinase/O(2)/NO-promoted reactions the total amounts of l-dopa and dopamine oxidized were significantly less than the amounts of the substrates oxidized by enzyme alone. These data indicate that the activity of the heme protein peroxidase was enhanced by NO, whereas tyrosinase, a copper-containing monoxygenase, was inhibited. The NO-mediated reduction of tyrosinase/O(2) activity may be attributed to the formation of an inhibitory copper.nitrosyl complex. An oxidized nitrodopamine derivative, considered to be either the quinone or semiquinone of 6-nitrosodopamine, was generated in peroxidase/H(2)O(2)/NO-mediated reactions with dopamine along with two oxidized melanin precursors, dopamine quinone and dopaminechrome. No corresponding nitroso compound was formed in reactions involving l-dopa or in any of the tyrosinase-mediated reactions. The formation of such a noncyclized nitrosodopamine represents an important alternative pathway in catecholamine metabolism, one that by-passes the formation of cytoprotective indole precursors of melanin. The results of this investigation suggest that cellular integrity and function can be adversely affected by NO-promoted oxidations of dopamine and other catechols, reactions that not only accelerate their conversion to reactive quinones but also form potentially cytotoxic noncyclized nitroso derivatives. Reduced levels of dopamine in the brain through NO-enhanced oxidation of the catecholamine will almost certainly be manifested by diminished levels of the dopamine-derived brain pigment neuromelanin.

Tobacco Mosaic Virus-Infected Tissue

Tobacco mosaic virus (TMV) infection has been studied in several investigations of Nicotiana tabacum leaf tissue. Earlier studies have suggested that TMV infection does not have precise infective selectivity vs. specific types of tissues. Also, such tissue conditions as vein banding, vein clearing, liquification and suberization may result from causes other than direct TMV infection. At the present time, it is thought that the plasmodesmata, ectodesmata and perhaps the plasmodesmata of the basal septum may represent the actual or more precise sites of TMV infection.TMV infection has been implicated in elevated levels of oxidative metabolism; also, TMV infection may have a major role in host resistance vs. concentration levels of phenolic-type enzymes. Therefore, enzymes such as polyphenol oxidase, peroxidase and phenylalamine ammonia-lyase may show an increase in activity in response to TMV infection. It has been reported that TMV infection may cause a decrease in o-dihydric phenols (chlorogenic acid) in some tissues.

Ultrastructural study of tobacco mosaic virus-infected chloroplasts

There have been several reports on the effects of tobacco mosaic virus (TMV) infection in Nicotiana tabacum leaf tissue. TMV infection does not appear to have an exact or precise infective selectivity, with respect to particular tissues. The tissue abnormalities such as lignification, suberization, vein banding, and vein clearing, may not be direct tissue responses to TMV infection. However, it has been suggested that plasmodesmata and possibly ectodesmata, may represent the actual sites of TMV infection, especially the plasmodesmata of the basal septum.TMV infection has also been implicated in host resistance vs. the levels of phenolic-type enzymes. A rise in oxidative metabolism has been associated with TMV infection, thus causing an increase in the, activity of peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase. In addition, TMV infection may cause a lowering of o-dihydric phenols such as chlorogenic acid in some tissues.

Distribution of Secondary Plant Metabolites and Their Biosynthetic Enzymes in Pea (Pisum sativum L.) Leaves

Leaves of a novel strain of peas (Pisum sativum L.) were used to determine the distribution of secondary metabolites and their biosynthetic enzymes. Leaf epidermal layers in this strain are easily separated from the parenchyma. Anthocyanins and flavonol glycosides were localized in epidermal vacuoles only. Among the biosynthetic enzymes studied, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), S-adenosyl-1-methionine (SAM):caffeic acid and SAM:quercetin methyltransferases (o-dihydric phenol methyltransferase, EC 2.1.1.42) and a flavonoid 7-O-glucosyltransferase (EC 2.4.1.91) were chiefly localized in the parenchyma, whereas trans-cinnamate 4-monooxygenase (EC 1.14.13.11), hydroxycinnamate:CoA ligases (EC 6.2.1.12) and a flavonoid 3-O-glucosyltransferase (EC 2.4.1.91) were found mainly in the epidermis. Flavanone (chalcone) synthase activity was found only in the epidermis, whereas chalcone isomerase (EC 5.5.1.6) was evenly distributed in epidermal and parenchyma tissues.

Two forms of O-methyltransferase in tobacco cell suspension culture

An S-adenosyl- l-methionine: o-dihydric phenol O-methyltransferase was isolated from tobacco cell suspension culture and was partially purified by (NH 4) 2SO 4 precipitation and successive chromatography on DEAE-Sepharose, Sephacryl S-200 and hydroxyapatite columns. It catalysed the O-methylation of 3 cinnamic acids, two coumarins and two flavonoids, but to different extents. Results obtained from polyacrylamide gel electrophoresis, m-/ p-methylation ratios and mixed substrate experiments indicated the existence of two forms of the enzyme which were resolved by chromatography on DEAE-cellulose. One form (MW 74000, pI 6.1, opt. pH 7.3) catalysed the meta-methylation of caffeic acid, while the other (MW 70000, pI 6.3, opt. pH 8.3) mediated the para-methylation of quercetin, though each form exhibited some activity against other substrates.

Biosynthesis of stizolobinic acid and stizolobic acid in higher plants.

Two new enzymes, which catalyze the synthesis of stizolobinic acid and stizolobic acid through oxidative ring cleavage of 3,4‐dihydroxyphenylalanine, subsequent recyclization and dehydrogenation of the resultant possible products were isolated from the soluble protein extract of Stizolobium hassjoo seedlings and were purified approximately 100‐fold by ammonium sulfate fractionation followed by column chromatography on Sephadex G‐100, DEAE‐cellulose and hydroxyapatite. The apparent molecular weights of both enzymes as determined by Sephadex G‐100 column chromatography were 45 000. The pH optima were 7.6 and 7.4, apparent Km values for 3,4‐dihydroxyphenylalanine as substrate were 1.67 mM and 1.39 mM for the enzymes to synthesize stizolobinic acid and stizolobic acid, respectively. Both enzymes required NADP+ for the activities. They were activated by Zn2+ and inhibited by sulfhydryl binding and zinc chelating agents. Both exhibited a strict specificity for 3,4‐dihydroxyphenylalanine as substrate and showed no affinities for other o‐dihydric phenols tested. The enzymes were very labile with a half‐life of 3–5 h at 30°C but they could be stored at −20°C for about 3 months under nitrogen gas with 0.1 mM dithiothreitol without appreciable loss of the catalytic enzyme activities.These enzymes have never been reported in any biological material. The trivial names, stizolobinic acid synthase and stizolobic acid synthase, are proposed.

Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures

Variations in teh activities of several enzymes of phenylpropanoid metabolism were studied in fermenter-grown cell suspension cultures of soyben ( Glycine max). Concomitant large increases and subsequent decreases in the activities of phenylalanine ammonina-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase, and two isoenzymes of p-coumarate:CoA ligase occurred prior to the stationary phase of the cell cultures. These findings represent a further example of an interdependent regulation of these enzymes of the general phenylpropanoid metabolism. The increases in all of these enzyme activities could be further enhanced by illunination of the cells. No comparable light effects and no significant changes were observed for the specific activity of an S-adenosylmethionine: o-dihydric phenol m- O-mehyltransferase and for the overall rate of the two-step reduction of feruloyl-CoA to coniferyl alcohol. These enzymatic reactions therefore appear to be regulated independently of the enzymes of the general phenylpropanoid metabolism.

Oxidation of catechol in plants: III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The K m for catechol was determined as 4 × 10 −4 m. The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Purification and properties of an o-dihydricphenol meta- O-methyltransferase from cell suspension cultures of parsley and its relation to flavonoid biosynthesis

1. 1. An O-methyltransferase directly related to flavone glycoside biosynthesis has been isolated from cell suspension cultures of parsley. The enzyme has been purified 82-fold by MnCl 2 and (NH 4) 2SO 4 precipitation and chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The enzyme catalyses the transfer of the methyl group of S-adenosyl- l-methionine to the meta position of o-dihydric phenols. 2. 2. Highest enzyme activities were obtained after illumination of the cells for 24 h with high intensities of white light from fluorescent lamps. 3. 3. The enzyme has a pH optimum around pH 9.7. It requires Mg 2+ and is not inhibited by p-chloromercuribenzoate or iodoacetamide. A molecular weight of about 48 000 was estimated from the elution volume after chromatography on a Sephadex G-100 column. 4. 4. Only o-dihydric phenols can function as substrates for the enzyme and only the meta-hydroxyl group is methylated. Luteolin (5,7,3′,4′-tetrahydroxyflavone) and its 7- O-glucoside with apparent K m values of 4.6·10 −5 and 3.1·10 −5 M, respectively, are the best substrates tested, whereas eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) ( K m = 1.2·10 −3M) and caffeic acid ( K m = 1.6·10 −3M) had much lower affinity for the enzyme. With luteolin as substrate the apparent K m value for S-adenosyl- l-methionine is 1.5·10 −4 M. 5. 5. Cell cultures also contain enzymatic activity for the synthesis of S-adenosyl- l-methionine from l-methionine and ATP. In contrast to the O-methyltransferase the activity of this enzyme is not influenced by illumination of the cultures.

The action of o-dihydric phenols in the hydroxylation of p-coumaric acid by a phenolase from leaves of spinach beet (Beta vulgaris L.).

1. Under defined conditions, the hydroxylation of p-coumaric acid catalysed by a phenolase from leaves of spinach beet (Beta vulgaris L.) was observed to develop its maximum rate only after a lag period. 2. By decreasing the reaction rate with lower enzyme concentrations or by increasing it with higher concentrations of reductants, the length of the lag period was inversely related to the maximum rate subsequently developed. 3. Low concentrations of caffeic acid or other o-dihydric phenols abolished this lag period. With caffeic acid, the rate of hydroxylation was independent of the reductant employed. 4. Hydroxylation was inhibited by diethyldithiocarbamate, but with low inhibitor concentrations hydroxylation recovered after a lag period. This lag could again be abolished by the addition of high concentrations of caffeic acid or other o-dihydric phenols. 5. Catechol oxidase activity showed no lag period, and did not recover from diethyldithiocarbamate inhibition. 6. The purified enzyme contained 0.17-0.33% copper; preparations with the highest specific activity were found to have the highest copper content. 7. The results are interpreted to suggest that the oxidation of o-dihydric phenols converts the enzymic copper into a species catalytically active in hydroxylation. This may represent the primary function for the catechol oxidase activity of the phenolase complex. The electron donors are concerned mainly, but not entirely, in the reduction of o-quinones produced in this reaction.

Biosynthesis of ethylene: Ethylene formation from methional by horseradish peroxidase

Ethylene is rapidly formed from methional by horseradish peroxidase, Mn +, SO 3 2−, and a specific phenol, or by horseradish peroxidase, SO 3 2−, catalytical amounts of hydrogen peroxide, and a specific phenol. The active phenols include some monohydric phenols and resorcinol. Ethylene formation is inhibited by o-dihydric phenols, Cu ++, or metal chelators. Ethylene formation accompanies the aerobic oxidation of sulfite during the reactions, and those inhibitors which inhibit ethylene formation also inhibit the enzymic oxidation of sulfite. A mechanism accounting for the formation of ethylene and the physiological significance of this reaction in relation to the biosynthesis of ethylene in plants is discussed.

The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.)

1. Polyphenol oxidase (EC 1. 10. 3.-) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mmu (E(1%) (1cm.), 13.5) and 611mmu (E(1%) (1cm.), 0.84) with a shoulder at 330mmu. The enzyme was bleached by substrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000+/-16000. The copper content was 0.32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 30 degrees . 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation of most substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation of the results to the mechanism of tea fermentation is discussed.

Anthranilate synthetase and the allosteric protein model.

Summary 1. A hypothesis is proposed to account for the observation that o-dihydric phenol synthesis by whole cells of the tryptophan auxotroph Aerobacter aerogenes NC3 is completely inhibited by low concentrations of tryptophan added to the substrates. It is claimed that in this strain a mutation has resulted in a defective anthranilate synthetase, such that the “active site” is changed, altering the enzymic function, but retaining allosteric inhibition by tryptophan. 2. In sustaining this hypothesis, previously unpublished results, evidence from a number of published sources arid work with Saccharomyces cerevisiae and Neurospora crassa, is considered in relation to (a) the original observations, (b) the normal enzymic site of feedback inhibition by tryptophan and (c) the nature of anthranilate synthetase. 3. As a result of considering various models for anthranilate synthetase in Aerobacter aerogenes and the inter-relationship between this activity and other tryptophan-path enzymic functions in Saccharomyces cerevisiae and Neurospora crassa, an allosteric aggregate enzyme model is proposed. It accounts for the genetic, nutritional and biochemical data. 4. Potential consequences of the aggregation of enzymic functions into alrosteric protein complexes are discussed and, in particular, an extension of the present concept of the function of allosteric proteins in biological systems.

A possible relationship between the formation of o-dihydric phenols and tryptophan biosynthesis by Aerobacter aerogenes

A number of auxotrophs of Aerobacter aerogenes requiring aromatic amino acids for growth have been shown to excrete o-dihydric phenols when incubated with a mixture of glucose, phosphate, magnesium and ammonium ions. Auxotrophs requiring histidine, methionine, cysteine or leucine do not excrete these substances under similar conditions. An auxotroph requiring tyrosine, phenylalanine and tryptophan for growth but unable to grow on shikimic acid accumulates a mixture of 3,4-dihydroxy-benzoic acid and catechol. A tryptophan auxotroph able to utilise anthranilic acid for growth accumulates 2,3-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid and catechol. A tyrosine auxotroph able to utilise 4-hydroxyphenylpyruvic acid has been shown to accumulate 2,3-dihydroxybenzoic acid and catechol. Catechol may arise consequent on 3,4-dihydroxybenzoic acid accumulation since suspensions of wild type A. aerogenes form catechol from 3,4-dihydroxybenzoic acid. Such suspensions also metabolise 3,4-dihydroxybenzoic acid and catechol to compounds not possessing the o-dihydric phenol structure when aerated in the absence of glucose. l-Tryptophan inhibits the accumulation of o-dihydric phenols by three different tryptophan auxotrophs of A. aerogenes but has no effect on the accumulation of these compounds by a tyrosine auxotroph or an auxotroph requiring tyrosine, phenylalanine and tryptophan for growth. The possible relationship of these o-dihydric phenols to the biosynthesis of aromatic compounds is discussed.

Role of oxidative metabolism in the localization of plant viruses

Upon infection, a rapid increase of polyphenoloxidase and peroxidase activity was found whenever host-virus combinations resulted in the formation of local lesions. The activation of phenol-oxidizing enzymes was slower and less in systemic hosts. The development of local lesions in Nicotiana glutinosa L. was accompanied by the decrease of o-dihydric phenols, mainly chlorogenic acid, in the host tissues. Histochemical tests suggested that, simultaneously, toxic quinones accumulate in the necrotic area. The production of quinones results in a rapid breakdown of added ascorbic acid in the tissues. Feeding of ascorbic acid or other reducing compounds (cysteine, glutathione) to the tissues in high concentrations markedly inhibited lesion formation, apparently by replacing the consumed endogenous reducing power and holding the phenols in a reduced state, and not by inhibiting the polyphenoloxidases. The development of local symptoms and the multiplication of viruses are not necessarily connected with each other since tobacco mosaic virus (TMV) did multiply in the ascorbic acid-treated N. glutinosa tissues without lesion formation. Around the lesions, in the healthy tissues, dehydrogenases are enormously activated and probably represent a defense reaction that helps to keep the phenolics in a reduced state. The role of polyphenol oxidation in lesion development is also shown by the relatively low activation of polyphenoloxidases in local lesion hosts upon exposure to 36° C, which results in a shift to systemic response.

On the nature and mode of action of the copperprotein, tyrosinase: II. Exchange experiments with radioactive copper and the functioning enzyme

1. 1. The exchange reaction between functioning tyrosinase and radiocupric ions has been studied. 2. 2. In general, high catecholase enzymes incorporated much more radioactive copper during catalysis of the oxidation of o-dihydric phenols than of monophenols. 3. 3. High cresolase enzymes retained little radioactivity in all experiments. 4. 4. The copper exchange data support the suggestion of two distinct activity sites in tyrosinase; i.e. catecholase and cresolase activity centers. It appears that the copper at cresolase activity sites is non-exchangeable. The magnitude of exchange of the copper atoms at catecholase activity sites seems to depend on the number of o-dihydric phenol molecules oxidized. 5. 5. The catecholase sites of tyrosinase appear to be little or not at all involved in the oxidation of monophenols. 6. 6. On the basis of the exchange experiments it is suggested that the oxidation of a monophenol by tyrosinase may not proceed via an o-dihydric phenol.