Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Isolation and characterization of catechol oxidase from Solanum melongena

Catechol oxidase was distributed in soluble and particulate fractions of Solanum melongena. The purified preparation appears to be homogeneous by polyacrylamide gel electrophoresis. The enzyme shows two pH maxima—with catechol, 6.5 and 7.5; and with dopa, 6.5 and 7.9. The latent form of the enzyme does not occur in S. melongena. The preparation resembles the enzyme from other sources in substrate specificity towards various mono- and diphenols, having a higher affinity for catechol than dopa; this tendency increases on purification. The cresolase activity decreases with purification and a lag period with p-cresol is observed. The oxidation of mono- and diphenols is inhibited by ascorbic acid, sulphydryl compounds and chelating agents.     

Properties of glutamate dehydrogenase from Lemna minor

Sterile cultures of Lemna minor grown in the presence of either nitrate, ammonium or amino acids failed to show significant changes in glutamate dehydrogenase (GDH) levels in response to nitrogen source. Crude and partially purified GDH preparations exhibit NADH and NADPH dependent activities. The ratio of these activities remain ca 12:1 during various treatments. Mixed substrate and product inhibition studies as well as electrophoretic behaviour suggest the existence of a single enzyme which is active in the presence of both coenzymes. GDH activity was found to be localized mainly in mitochondria. Kinetic studies revealed normal Michaelis kinetics with most substrates but showed deviations with NADPH and glutamate. A Hill-coefficient of 1.9 determined with NADPH indicates positive cooperative interactions, whereas a Hill-coefficient of 0.75 found with glutamate may be interpreted in terms of negative cooperative interactions. NADH dependent activity decreases rapidly during gel filtration whereas the NAD⁺ and NADPH activities remain unchanged. GDH preparations which have been pretreated with EDTA show almost complete loss of NADH and NAD⁺ activities. NADPH activity again remains unaffected. NAD⁺ activity is fully restored by adding Ca²⁺ or Mg²⁺, whereas the NADH activity can only be recovered by Ca²⁺ but not at all by Mg²⁺. Moderate inhibition of GDH reactions observed with various adenylates are fully reversed by adding Ca²⁺, indicating that the adenylate inhibition is due solely to the chelating properties of these compounds.     

Purification and properties of alcohol oxidase from Tanacetum vulgare

Alcohol oxidase (alcohol: O₂ oxidoreductase) from leaves of Tanacetum vulgare has been purified 5150-fold to homogeneity on disc electrophoresis and gel electrofocussing. The enzyme which is probably flavoprotein, has molecular weight 180 000 daltons and is comprised of two sub-units of 94 000 and 75 000 daltons. It is active over a broad range (pH 5–9) and best accepts primary aliphatic alcohols with 6 to 10 carbons, especially those with a 2-ene group. K_m values for hex-trans-2-ene-1-ol, geraniol (3,7-dimethylocta-trans-2,6-dien-1-ol) and n-octanol were 0.19, 1.56 and 0.49 mM respectively. The significance of the enzyme in the formation of leaf aldehyde (hex-trans-2-ene-1-al) and in terpene metabolism is discussed.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

2,3-dihydrojaborosalactone A,a withanolide from Acnistus breviflorus

From Acnistus breviflorus the new 27-hydroxy-5β,6β-epoxy-1-oxo-(22R)-witha-24-enolide (2,3-dihydrojaborosalactone A) as well as seven known withanolides, withaferin A, 2,3-dihydrowithaferin A, 6α-chloro-5β-hydroxywithaferin A, 5,6-deoxywithaferin A, jaborosalactone A, jaborosalactone D and jaborosalactone E were isolated and characterized by means of spectroscopic (¹H NMR, ¹³ C NMR and mass spectral) methods. Depending on the extraction solvent (methanol or ethanol), a known artifact (3β-methoxy-2,3-dihydrowithaferin A) and the new 5α-methoxy-4,5-dihydrojaborosalactone B and 5α-ethoxy-4,5-dihydrojaborosalactone B were also isolated and characterized.     

Properties of the solvent-stimulated ATPase activity of chloroplast coupling factor 1 from Chlamydomonas reinhardii

The effects of solvents on the ATPase activity of chloroplast coupling factor 1 (CF₁) isolated from wild-type Chlamydomonas reinhardii have been studied. Of the solvents examined, the following order summarizes their maximal ability to stimulate the ATPase activity of CF₁: ethanol > methanol>allyl alcohol >n-propanol > acetone≈dioxane > ethylene glycol. Glycerol inhibits the CF₁ activity at all concentrations. In the absence of organic solvents, 50% of the activity of the enzyme is irreversibly lost after a 10 min incubation at 65–70°C. Ethanol (23%) causes a 30°C drop in the temperature required for 50% inactivation. ATP partially stabilizes the CF₁ in the presence, but not in the absence, of ethanol. In the absence of organic solvents, both free Mg²⁺ and ADP inhibit the CF₁-ATPase. Mg²⁺ is a noncompetitive inhibitor with respect to MgATP, and the kinetic constants are: V, 6.3 μmol ATP hydrolyzed/mg protein per min; K_m(MgATP), 0.23 mM; K_ii(Mg²⁺), 27 μM; and K_is(Mg²⁺), 50 μM. In the presence of ethanol, double-reciprocal plots are no longer linear and have a Hill coefficient of about 1.8±0.1. V increases about 10–12-fold. The pattern of inhibition by Mg²⁺ appears to change from noncompetitive to competitive with respect to MgATP. In addition, ADP no longer inhibits the MgATPase activity of CF₁.     

Cellular and chloroplast lipid composition of the leaves of Mimosa pudica

Cellular and chloroplast lipids of the leaves of Mimosa pudica have been analysed. Qualitatively the total lipid composition of this plant is similar to that reported for the photosynthetic tissues of other plants. Chloroplast lipids show some resemblance to those of algae. The cerebroside fraction of both leaves and chloroplasts contains a polyunsaturated fatty acid (20:4ω3) and a long chain sphingosine base whose R_f value coincides with that from ox brain cerebroside and not with that of phytosphingosine from spinach.     

An aminopeptidase from Agave americana thermodynamic studies

Purified Agave aminopeptidase was characterized with respect to the thermodynamic properties of the reaction catalysed by the enzyme. Kinetic studies were conducted at different temperatures ranging from 7.8 to 45°. The energy of activation, E_a, as well as the constants ΔH, ΔF and ΔS were calculated for both the formation of the enzyme-substrate complex, and the dissociation of the enzyme-product complex. Kinetic studies in buffers with varying dielectric constants enabled the determination of the electrostatic as well as the non-electrostatic components of ΔS. These results fit well into the overall kinetic picture of this enzyme-catalysed reaction as reported previously.     

Mono- and diphenolase activity from fruit of Malus pumila

Apple fruit used for beverage production has a polyphenol oxidase which does not hydroxylate tyrosine under any conditions but it hydroxylates p-coumaric acid in the presence of NADH, and phloridzin in the absence of cofactors. The apparent K_ms for hydroxylation of phloridzin and p-coumaric acid are 1.5 and 4 mM, respectively. However, subsequent oxidation of 3-hydroxyphloridzin or caffeic acid has an apparent K_m of 200 nM. The oxidation products of 3-hydroxyphloridzin are complex and a stable dimeric quinone is finally formed. The apparent K_ms for oxidation of catechin, epicatechin, chlorogenic acid, l-Dopa and 4-methyl catechol are 4.7, 5.7, 6.0, 2.7 and 3.2 mM, respectively. The K_m for oxygen was 4.3 % although there was marked substrate inhibition by oxygen above 30 %. Polyphenol oxidase was stable at pH 3.5–4.5 with an optimum of 4.5.     

The phenolics and a hydrolysable tannin polyphenol oxidase of Medinilla magnifica

The production of viable meristem cultures of Medinilla magnifica has proved to be very difficult. This may be due, in part, to a pronounced ‘browning’ response of the tissues on cutting. For this reason the phenolic compounds and the hydrolysable-tannin polyphenol oxidase from Medinilla were studied. The distribution of the compounds was: simple phenols 19% , flavonoids 5% , hydrolysable tannins 69% , condensed tannins 7%. Amongst the simple phenols and phenolic acids, the following were identified: phloroglucinol, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, gallic acid (both in free and bound form the most abundant simple phenol), syringic acid, trans-p-coumaric acid, trans-ferulic acid and trans-caffeic acid. No kaempferol or quercetin or their derivatives were detected but condensed tannins are present. Methods for the extraction, fractionation and quantitative determination of phloroglucinol and the phenolic acids, as well as correction factors for losses during the extraction, alkali treatment and derivatization, are presented in a supplementary publication. With regard to the hydrolysable tannin polyphenol oxidase activity of Medinilla stems, the enzyme(s) is rather specific since at neither of its two pH optima (6 and 7) could a classical polyphenol oxidase activity be detected. The enzyme was strongly inhibited by 2-mercaptoethanol. Preliminary experiments have further shown that in addition to the hydrolysable tannins of the tissue, the ferrous ions of the medium, and oxygen together with the hydrolysable tannin polyphenol oxidase could play a role in the browning response. Ways to overcome this difficulty have been suggested.     

Induction of glycolate oxidase by SO2 in Nicotiana tabacum

In contrast to the inhibitory action of sulfite on glycolate oxidase, the specific activity of the enzyme in tobacco leaves exposed to SO₂ for 18 hr increases in proportion to the SO₂ concentration. This increase is strongly reduced by pretreatment with cycloheximide. As a consequence of induced de novo synthesis of glycolate oxidase the glycolate content of the leaves is markedly reduced after 18 hr exposure to SO₂.     

Phenolic oxidase activities in glandular trichomes of Solanum berthaultii

Polyphenol oxidase and peroxidase activities were detected in foliar glandular trichomes of the wild, insect-resistant potato species, Solanum berthaultii. These enzyme activities may provide the basis for conversion of clear, viscous trichome exudate into a hard, brown substance which is formed rapidly after insect attack.     

Properties of purified chloroplastic and cytoplasmicvalyl- and leucyl-tRNA synthetases from Euglena gracilis

The catalytic properties of purified chloroplastic and cytoplasmic valyl- and leucyl-tRNA synthetasesfrom Euglena gracilis were studied and compare     

Chloroplast cysteine synthases of Trifolium repens and Pisum sativum

About 68–86% of the cysteine synthase activity in leaf tissue of white clover (Trifolium repens) and peas (Pisum sativum cultivar Massey Gem) was associated with chloroplasts. The enzymes from white clover and peas were purified ca 66 and 12-fold respectively. For clover, the K_m values determined by calorimetric and S^2? ion electrode methods were: S^2? 0.51 and 0.13 mM; O-acetylserine (OAS), 3.5 and 2.O mM respectively. The analogous values for the pea enzyme were: S^2?, 0.24 and 0.06 mM; OAS, 3.1 and 0.24 mM. Both enzymes were inhibited by cystathionine and cysteine. Pretreatment with cysteine inactivated the enzyme, but addition of pyridoxal phosphate caused partial reactivation. Isolated pea chloroplasts (70–75 % intact) catalysed OAS-dependent assimilation of sulphide at a mean rate of 88 μmol/mg Chl/hr. About 85 % of the OAS-dependent sulphide assimilated was recovered as cysteine. The rates were unaffected by light and 2 μM DCMU. Sonicating the chloroplasts enhanced the rate by 1.3–2 fold. Cysteine synthase activity was associated with the chloroplast stroma. Similar results were obtained for clover chloroplasts except that both the intactness and the rates were lower.     

Partial characterization of a phenolic pigment from sporocarps of Phellinus igniarius

A dark brown phenolic polymer, isolated from mature sporocarps of Phellinus igniarius (DC. ex Fr.) Quél., is responsible for the darkening of sporocarp tissue when a drop of base is applied. The pigment is rich in aliphatic as well as phenolic hydroxyl groups, and also contains significant numbers of carboxyls and carbonyls. The presence of 3,4-dihydroxyphenyl moieties was documented by the identification of veratric and metahemipinic acids from oxidative degradation of methylated samples. Despite a 2% methoxyl content, the polymer contains no guaiacyl or syringyl moieties and therefore contains no lignin. It appears to be formed by the oxidative polymerization of a molecule containing a 3,4-dihydroxystyryl moiety. Hispidin was identified as its trimethyl ether in methylated extracts of pigmented mycelial mats of Phellinus igniarius grown in liquid culture.     

Purification and properties of indole 2,3-dioxygenase from maize leaves

An indole 2,3-dioxygenase was purified ca 38-fold from maize leaves. The enzyme had an MW of about 98000, an optimum pH of 5.0 and the energy of activation was 9.1 kcal/mol. The K_max for indole was 1.4 × 10^?4 M. The enzyme was inhibited by diethyldithiocarbamate, salicylaldoxime and sodium dithionite. The inhibition by diethyldithiocarbamate was specifically reversed by Cu²⁺. The dialysed enzyme was stimulated by Cu²⁺. Four atoms of oxygen were utilized in the disappearance of 1 mole of indole. Inhibition of the enzyme by -SH compounds and -SH group inhibitors, and their partial removal by Cu²⁺ only, suggested the involvement of -SH groups in binding of Cu²⁺ at the catalytic site.     

Purification and some properties of polyphenol oxidase from the yam tubers, Dioscorea bulbifera

In a comparison of the polyphenol oxidase activity of various species of yam tubers the greatest enzyme activity was found in D. bulbifera. The enzyme was purified from acetone powder extracts of this plant. Ammonium sulphate fractionation, followed by ion exchange chromatography and gel filtration gave 22-fold purification. The final product gave a single band on polyacrylamide disc gel electrophoresis. The purified enzyme showed activity towards catechol, pyrogallol and dl-β-3,4-dihydroxyphenylalanine (dl-DOPA) and had a MW 115000 ± 2000. It was characterized by response to various inhibitors. β-Mercaptoethanol, dithioerythritol, l-cysteine, sodium metabisulphite and KCN inhibited strongly.     

Quantitative analysis of anticytokinin activity of 4-substituted-2-methylthiopyrido[2,3-d]pyrimidines

Ten 4-substituted-2-methylthiopyrido[2,3-d]pyrimidines were synthesized and tested for their cytokinin-antagonistic activity by the tobacco callus bioassay. This series of compounds constitutes the first example of anti-cytokinins which possess a fused 6-6 membered ring system. The treatment of Lineweaver and Burk, the method of classical enzyme kinetics, revealed competitive inhibition of cytokinin-induced tobacco callus growth. The variation of activity with the systematic transformation of 4-substituents was analysed quantitatively with physicochemical substituent parameters and regression analysis. The results indicated the predominant importance of substituent width for binding of the antagonists at the receptor site of cytokinins.     

Ingenol esters from the pro-inflammatory fraction of Euphorbia kamerunica

A series of unstable mono- and di-esters of the tetracyclic diterpene ingenol were isolated from the pro-inflammatory ether-soluble fraction of the latex of Euphorbia kamerunica. The esters were isolated by a neutral process involving column and thin-layer chromatography. The monoesters were identified by spectroscopic methods and hydrolysis reactions as ingenol-3-decanoate, ingenol-3-dodecanoate, ingenol-5-hexadienoate and ingenol-5-octenoate and the diesters as 20-acetyl-ingenol-3-octenoate and 20-acetyl-ingenol-3-angelate.