Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H₂O₂, and NH₃. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220?kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32?kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K _m values for benzylamine and phenethylamine were 2.7?×?10^?4 and 7.1?×?10^?4?M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3???M. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2?mol of FAD per 220,000?g of enzyme.     

New quinoxalinone inhibitors targeting secreted phospholipase A2 and α-glucosidase

Elevated blood glucose and increased activities of secreted phospholipase A2 (sPLA2) are strongly linked to coronary heart disease. In this report, our goal was to develop small heterocyclic compound that inhibit sPLA2. The title compounds were also tested against α-glucosidase and α-amylase. This array of enzymes was selected due to their implication in blood glucose regulation and diabetic cardiovascular complications. Therefore, two distinct series of quinoxalinone derivatives were synthesised; 3-[N′-(substituted-benzylidene)-hydrazino]-1H-quinoxalin-2-ones 3a–f and 1-(substituted-phenyl)-5H-[1,2,4]triazolo[4,3-a]quinoxalin-4-ones 4a–f. Four compounds showed promising enzyme inhibitory effect, compounds 3f and 4b–d potently inhibited the catalytic activities of all of the studied proinflammatory sPLA2. Compound 3e inhibited α-glucosidase (IC₅₀?=?9.99?±?0.18 µM); which is comparable to quercetin (IC₅₀?=?9.93?±?0.66 µM), a known inhibitor of this enzyme. Unfortunately, all compounds showed weak activity against α-amylase (IC₅₀?>?200 µM). Structure-based molecular modelling tools were utilised to rationalise the SAR compared to co-crystal structures with sPLA2-GX as well as α-glucosidase. This report introduces novel compounds with dual activities on biochemically unrelated enzymes mutually involved in diabetes and its complications.     

Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source

1. The yeast Candida boidinii was grown on glucose as carbon source with a range of amines and amino acids as nitrogen sources. Cells grown on amines contained elevated activities of catalase. If the amines contained N-methyl groups, formaldehyde dehydrogenase, formate dehydrogenase and S-formylglutathione hydrolase were also elevated in activity compared with cells grown on (NH₄)₂SO₄. 2. Cells grown on all the amines tested, but not those grown on urea or amino acids, contained an oxidase attacking primary amines, which is referred to as methylamine oxidase. In addition, cells grown on some amines contained a second amine oxidase, which is referred to as benzylamine oxidase. 3. Both amine oxidases were purified to near homogeneity. 4. Benzylamine oxidase was considerably more stable at 45 and 50°C than was methylamine oxidase. 5. Both enzymes had a pH optimum in the region of 7.0, and had a considerable number of substrates in common. There were, however, significant differences in the substrate specificity of the two enzymes. The ratio V/K^app._m increased with increasing n-alkyl carbon chain length for benzylamine oxidase, but decreased for methylamine oxidase. 6. Both enzymes showed similar sensitivity to carbonyl-group reagents, copper-chelating agents and other typical `diamine oxidase inhibitors'. 7. The stoicheiometry for the reaction catalysed by each enzyme was established. 8. The kinetics of methylamine oxidase were examined by varying the methylamine and oxygen concentrations in turn. A non-Ping Pong kinetic pattern with intersecting double-reciprocal plots was obtained, giving K_m values of 10μm for O₂ and 198μm for methylamine. The significance of this unusual kinetic behaviour is discussed. Similar experiments were not possible with the benzylamine oxidase, because it seemed to have an even lower K_m for O₂. 9. Both enzymes had similar subunit M_r values of about 80000, but the benzylamine oxidase behaved as if it were usually a dimer, M_r 136000, which under certain conditions aggregated to a tetramer, M_r 288000. Methylamine oxidase was mainly in the form of an octamer, M_r 510000, which gave rise quite readily to dimers of M_r 150000, and on gel filtration behaved as if the M_r was 286000.     

Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Amine Oxidases of Microorganisms

With the crystalline preparations of amine oxidase of Aspergillus niger, some properties of the enzyme were investigated. The enzyme was stable in phosphate buffer of pH over the range of 6.0 to 7.0. On heating, the enzyme was stable up to 35°C, but, above 40°C, it was rapidly destroyed.

The recrystallized enzyme was at least 90% pure when examined in the ultracentrifuge. The molecular weight was determined to be approximately 252,000. The enzyme was pink in color and shown an absorption maximum at about 480 m/μ. This absorption maximum was abolished by substrates as well as by sodium dithionite, and was restored by oxygenation.

The enzyme oxidized preferentially aliphatic monoamines of C₃—C₆. The other monoamines, such as benzylamine, phenethylamine, histamine and agmatine, were oxidized as well. The aliphatic diamines of C₄—C₆ were oxidized but in rather low rates. The rates of oxidation showed optima at pH 7.5, 7.2, 7.8 and 8.6 for n-butylamine, benzylamine, histamine and putrescine, respectively.     

VEGFR-2 inhibitors and apoptosis inducers: synthesis and molecular design of new benzo[g]quinazolin bearing benzenesulfonamide moiety

Two series of novel 4-(2-(2-(2-(substituted) hydrazinyl)-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 5–17 and 4-(2-(2-(substituted-1H-pyrazol-1-yl)-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 18–24 were synthesised from the starting material 4-(2-(2-hydrazinyl-2-oxoethylthio)-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 5, to be evaluated for their inhibitory activity towards VEGFR-2. The target compounds 5–24, were screened for their cytotoxic activity against MCF-7 breast cancer cell line and the percentage inhibition against VEGFR-2. Compounds 9, 20, 22 and 23, showed excellent VEGFR-2 inhibitory activity with IC₅₀ ranging from 0.64 to 1.04?µm. Being the most potent, compound 9 was evaluated for its apoptotic inducer effect by studying the effect on caspase-3, it was found to increase its level. Compound 9 boosted the level of Bax and reduced the level of BCl2, compared to the control. Cell cycle analysis was conducted, compound 9 showed cell cycle arrest at G2/M phase. Moreover, mild cytotoxic effect (IC₅₀?=?29.41?µm, respectively) in normal breast cells MCF-12?A, was observed when treated with the same compound. Finally, a molecular docking study was performed to investigate the possible binding interaction inside the active site of the VEGFR-2 enzyme.     

Structure–activity relationship studies of 3-dodecanoylindole-2-carboxylic acid inhibitors of cytosolic phospholipase A2α-mediated arachidonic acid release in intact platelets: variation of the keto moiety

Recently we found that 1-methyldodecanoylindole-2-carboxylic acid (1) and 1-[2-(4-carboxyphenoxy)ethyl]-3-dodecanoylindole-2-carboxylic acid (4) were inhibitors of the cytosolic phospholipase A₂α (cPLA₂α)-mediated arachidonic acid release in calcium ionophore A23187-stimulated human platelets with IC₅₀-values of 4.8 μM (1) and 0.86 μM (4). We have now replaced the 3-acyl residue of these compounds by alkylated sulfinyl-, sulfony-, sulfinamoyl-, sulfamoyl-, carbonylamino-, or carbonylaminomethyl-substituents. Structure–activity relationship studies revealed that the pronounced cellular activity of 4 strongly depends on the presence of the 3-acyl moiety. Surprisingly, when testing 4 and its derivatives in an assay with the isolated cPLA₂, none of these compounds showed an inhibitory potency at 10 μM indicating that they do not inhibit cPLA₂α in the cells by a direct interaction with the active site of the enzyme.     

Aerobic and Anaerobic Catabolism of Vanillic Acid and Some Other Methoxy-Aromatic Compounds by Pseudomonas sp. Strain PN-1

Vanillic acid (4-hydroxy-3-methoxybenzoic acid) supported the anaerobic (nitrate respiration) but not the aerobic growth of Pseudomonas sp. strain PN-1. Cells grown anaerobically on vanillate oxidized vanillate, p-hydroxybenzoate, and protocatechuic acid (3,4-dihydroxybenzoic acid) with O₂ or nitrate. Veratric acid (3,4-dimethoxybenzoic acid) but not isovanillic acid (3-hydroxy-4-methoxybenzoic acid) induced cells for the oxic and anoxic utilization of vanillate, and protocatechuate was detected as an intermediate of vanillate breakdown under either condition. Aerobic catabolism of protocatechuate proceeded via 4,5-meta cleavage, whereas anaerobically it was probably dehydroxylated to benzoic acid. Formaldehyde was identified as a product of aerobic demethylation, indicating a monooxygenase mechanism, but was not detected during anaerobic demethylation. The aerobic and anaerobic systems had similar but not identical substrate specificities. Both utilized m-anisic acid (3-methoxybenzoic acid) and veratrate but not o- or p-anisate and isovanillate. Syringic acid (4-hydroxy-3,5-dimethoxybenzoic acid), 3-O-methylgallic acid (3-methoxy-4,5-dihydroxybenzoic acid), and 3,5-dimethoxybenzoic acid were attacked under either condition, and formaldehyde was liberated from these substrates in the presence of O₂. The anaerobic demethylating system but not the aerobic enzyme was also active upon guaiacol (2-methoxyphenol), ferulic acid (3-[4-hydroxy-3-methoxyphenyl]-2-propenoic acid), 3,4,5-trimethoxycinnamic acid (3-[3,4,5-trimethoxyphenyl]-2-propenoic acid), and 3,4,5-trimethoxybenzoic acid. The broad specificity of the anaerobic demethylation system suggests that it probably is significant in the degradation of lignoaromatic molecules in anaerobic environments.     

Potent inhibitory effects of benzyl and p-xylidine-bis dithiocarbamate sodium salts on activities of mushroom tyrosinase

A novel monofunctional benzyldithiocarbamate, C₆H₅CH₂NHCSSNa (I), and a bifunctional p-xylidine-bis(dithiocarbamate), NaSSCNHCH₂C₆H₄CH₂NHCSSNa (II), as sodium salts, were synthesized by reaction between p-xylylenediamine or benzylamine with CS₂ in the presence of NaOH. They were characterized by spectroscopic techniques such as ¹H NMR, IR, and elemental analysis. These water-soluble compounds were examined for their inhibition of both activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. l-3,4- Dihydroxyphenylalanine (L-DOPA) and l-tyrosine were used as natural substrates for the catecholase and cresolase enzyme reactions, respectively. Kinetic studies showed noncompetitive inhibition of I and mixed type inhibition of II on both activities of MT. The inhibition constant (K_I) of II was smaller than that of I. Raising the temperature from 27 to 37°C caused a decrease in K_I values of I and an increase in values of II. The binding process for inhibition of I was only entropy driven, which means that the predominant interaction in the active site of the enzyme is hydrophobic; meanwhile, the electrostatic interaction can be important for the inhibition of II due to the enthalpy driven binding process. Fluorescence studies showed a decrease of emission intensity without a shift of emission maximum in the presence of different concentrations of compounds. An extrinsic fluorescence study did not show any considerable change of the tertiary structure of MT. Probably, the conformation of inhibitor-bound MT is stable and inflexible compared with uninhibited MT.     

Pyruvate fermentation in light-grown cells ofRhodospirillum rubrum during adaptation to anaerobic dark conditions

Pyruvate fermentation inRhodospirillum rubrum (strains F₁, S₁, and Ha) was investigated using cells precultured on different substrates anaerobically in the light and than transferred to anaerobic dark conditions. Pyruvate formate lyase was always the key enzyme in pyruvate fermentation but its activity was lower than in cells which have been precultured aerobically in darkness. The preculture substrate also had a clear influence on the pyruvate formate lyase activity. Strains F₁ and S₁ metabolized the produced formate further to H₂ and CO₂. A slight production of CO₂ from pyruvate, without additional H₂-production, could also be detected. It was concluded from this that under anaerobic dark conditions a pyruvate dehydrogenase was also functioning. On inhibition of pyruvate formate lyase the main part of pyruvate breakdown was taken over by pyruvate dehydrogenase.When enzyme synthesis was inhibited by chloramphenicol, propionate production in contrast to formate production was not affected. Protein synthesis was not significant during anaerobic dark culture. Bacteriochlorophyll. however, showed, after a lag phase, a clear rise.Abbreviations Bchl Bacteriochlorophyll - CoA Coenzyme A - DSM Deutsche Sammlung von Mikroorganismen (Göttingen) - OD optical density - PHBA poly--hydroxybutyric acid - R Rhodospirillum     

Attachment Behavior of Shewanella putrefaciens onto Magnetite under Aerobic and Anaerobic Conditions

In this study, batch experiments were used to characterize attachment behavior of Shewanella putrefaciens strain 200R to ferrihydrite and magnetite. Attachment was quantified in batch experiments with a 0.01 M NaNO ₃ solution as a function of pH (ranging from 3 to 10), sorbed anion (PO₄ ^{3 ?} ), and growth conditions (aerobic vs. anaerobic). Electrophoretic mobility data was collected for S. putrefaciens cells and magnetite grains and used as a means to interpret the role of electrostatic interaction in attachment studies. Little difference in attachment behavior was observed as a function of growth conditions or surface treatments. The exception was at pH ranging from 2 to 4, under anaerobic conditions, where increased attachment was measured on magnetite surfaces with sorbed PO₄ ^{3 ?} . This increased attachment was attributed to development of Fe-PO₄ surface complexes or secondary mineral phases, resulting in altered surface interactions between cell and mineral surfaces. Attachment was irreversible and increased with time under anaerobic conditions even under elevated pH conditions unfavourable to electrostatic interactions between cells and mineral surfaces. These results suggest that electrophoretic mobility data in this system is not a good predictor of attachment behavior, while surface charge development via protonation and deprotonation of surface functional groups is consistent with experimental attachment data. In this study, S. putrefaciens appears to utilize polymers or pili to remain attached to Fe-oxides and this process may facilitate Fe reduction on these surfaces. Results from this study underscore the need for quantitative bulk measurements of microbial attachment to accurately predict partitioning of dissimilatory iron reducing bacteria between solution and solid phases.     

The effect of managing nutrients in the performance of anaerobic digesters of municipal wastewater treatment plants

Is it possible to create conditions in the anaerobic digesters to control nutrients without changing the performance of a reactor? This study investigates an answer for this question. To this purpose, anaerobic reactors are operated at high concentrations of Mg²⁺ ion to harvest the nutrient ions (NH₄ ⁺ and PO₄ ^3?) in the form of struvite, that is, magnesium ammonium phosphate. The effects of this modification on the anaerobic digestion of sewage sludge were investigated in terms of chemical oxygen demand (COD) removal and cumulative CH₄ production as well as the changes in the biological diversity. The results showed that approximately 50 % of the nutrients (NH₄ ⁺ and PO₄ ^3?) were removed regardless of the method adopted for the addition of Mg²⁺ ion, slug or daily dosing. The numbers of Methanosaeta and Methanosarcina in the samples withdrawn prior to and after the addition of Mg²⁺ did not show significant difference according to the results obtained from qPCR analyses. The research results showed that the addition of Mg²⁺ into the anaerobic digesters in municipal wastewater treatment facilities may help to remove the nutrients from the effluent while recovering in their solid forms.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C₃H₇OCS₂Na (I), C₄H₉OCS₂Na (II) and C₅H₁₁OCS₂Na (III), were synthesized, by the reaction between CS₂ with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K_i values of 9.8, 7.2 and 6.1 μM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 μM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (α>1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S^? ) of the anion xanthate, which leads to similar values of K_i for all three xanthates. Different K_i values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

Antifouling activity of sponge-derived polybrominated diphenyl ethers and synthetic analogues

The antifouling (AF) activity of 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)- 6-methylbenozoic acid methyl ester (1), 3,5-dibromo-2-(2′,4′-dibromophenoxy)phenol (2); 3,4,5-tribromo-2-(2′,4′-dibromophenoxy)phenol (3), 3,4,5-tribromo-2-(2′-bromophenoxy)phenol (4), 3,5-dibromo-2(2′,4′-dibromophenoxy)phenol (5), 3,4,5,6-tetrabromo-2-(2′-bromophenoxy)phenol (6); 4-phenoxyphenol (7), 4-phenoxyaniline (9), 1-chloro-4-phenoxybenzene (10); 1-bromo-4-phenoxybenzene (13) was investigated against marine bacteria, a diatom, barnacle larvae and mussel juveniles. The naturally occurring compound 2 showed the strongest AF activity in all bioassays but lacked toxicity. It inhibited the growth of all tested bacterial strains (MIC = 0.02 – 1.52 μM) and its 50% effective concentrations (EC₅₀) were 0.24 μM (diatom test), 0.66 μM (mussel test) and 1.26 μM (barnacle test). Among the commercially available derivates, compound 7 was the most active in bacterial and diatom bioassays but its activity was lower than that of compound 2. Overall, the naturally occurring compounds showed stronger activity than the commercially available analogues and could be possible future non-toxic AF candidates.     

Carbonic anhydrase inhibition. Insight into the characteristics of zonisamide,topiramate, and the sulfamide cognate of topiramate

Some useful therapeutic agents inhibit certain carbonic anhydrase (CA) isozymes to varying degrees. We have conducted enzyme kinetics studies in a 4-nitrophenyl acetate (4-NPA) hydrolysis assay with the marketed antiepileptic drugs topiramate (1) and zonisamide (2) to determine if their full inhibition of human CA-II and CA-I requires extended preincubation conditions. We found that neither 1 nor 2 requires appreciable preincubation with either enzyme to manifest full inhibitory activity. We also examined the sulfamide cognate of topiramate (3) to characterize its CA inhibitory activity, and confirmed that it is a very weak inhibitor, unlike 1 or 2. In a CO₂ hydration assay, 3 behaved as a very weak, partial inhibitor of CA-II and CA-I. We conclude that topiramate (1), zonisamide (2), and sulfamide 3 do not require extended exposure to human CA-I or CA-II to manifest full inhibitory activity (4-NPA assay).     

A catalytic mechanism for the enzyme benzylamine oxidase from pig plasma

Initial-velocity and product-inhibition studies on the enzyme benzylamine oxidase from pig plasma indicate that the order of substrate addition and product release is benzylamine on, ammonia off, oxygen on, hydrogen peroxide off, benzaldehyde off. Ammonia, but not benzaldehyde, is released under strictly anaerobic conditions which provides independent evidence for this order. Benzyl alcohol is a substrate for the enzyme. A chemical mechanism consistent with all the data is proposed.     

Benzo[g]quinazolin-based scaffold derivatives as dual EGFR/HER2 inhibitors

Targeting EGFR has proven to be beneficial in the treatment of several types of solid tumours. So, a series of novel 2-(4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydrobenzo[g]quinazolin-2-ylthio)-N-substituted acetamide 5–19 were synthesised from the starting material 4-(2-mercapto-4-oxobenzo[g]quinazolin-3(4H)-yl) benzenesulfonamide 4, to be evaluated as dual EGFR/HER2 inhibitors. The target compounds 5–19, were screened for their cytotoxic activity against A549 lung cancer cell line. The percentage inhibition of EGFR enzyme was measured and compared with erlotinib as the reference drug. Compounds 6, 8, 10, and 16 showed excellent EGFR inhibitory activity and were further selected for screening as dual EGFR/HER2 inhibitors. The four selected compounds showed IC₅₀ ranging from 0.009 to 0.026?µM for EGFR and 0.021 to 0.069?µM for the HER2 enzyme. Compound 8 was found to be the most potent in this study with IC₅₀ 0.009 and 0.021?µM for EGFR and HER2, respectively.     

Gas exchange of two CAM species of the genus Cissus (vitaceae) differing in morphological features

NADP-malate dehydrogenase extracted from darkened leaves of the C₃ plants pea, barley, wheat and spinach was activated by reduced glutathione, a monothiol, as well as by dithiothreitol (DTT). However, in the C₄ plants maize and Flaveria trinervia, only dithiothreitol could effectively activate the enzyme. There was no activation of the maize enzyme and little or no activation of the F. trinervia enzyme by glutathione. The failure of glutathione to activate NADP-MDH in leaf extracts of maize and F. trinervia may indicate there is some difference in disulfide groups of the protein compared to the C₃ plant enzyme. Both DTT and glutathione could activate NADP-malate dehydrogenase in a partially purified enzyme preparation from pea leaves with or without addition of partially purified thioredoxin. However, the required concentration of reductant was lower with addition of thioredoxin than in its absence. In extracts of C₃ species and the partially purified pea enzyme the level of activation after 40 to 60 min under aerobic conditions was higher (up to twofold) with DTT than with glutathione. Under anaerobic conditions, the initial rate of activation was about twice as high with DTT as with glutathione, but the total activation after 40 to 60 min was similar. Ascorbate was totally ineffective as a reducing agent in activating NADP-MDH from C₃ or C₄ plants, possibly due to its more positive redox potential.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - GSH Reduced Glutathione - NADP-MDH NADP-malate Dehydrogenase     

On the acceptor specificity of glycollate oxidase of Nicotiana tabacum

Summary The effect of compounds on the activity of ammonium sulphate preparations of glycollate oxidase from Nicotiana tabacum cv. John Williams' Broadleaf and the aurea mutant Su/su is reported. Coupling to DCPIP as terminal oxidant under anaerobic conditions gave greater rates of glycollate oxidation than when measured as O₂ uptake in the presence of cyanide. The enzyme also linked to DCPIP in the presence of O₂, showing that it is a facultative aerobic dehydrogenase. Catalytic amounts of PMS stimulated enzyme-dependent oxygen uptake and DCPIP reduction under aerobic and anaerobic conditions. This further suggests that an intermediate carrier, or alternate acceptor, depending on concentration, exists before O₂ in vivo. Naturally occurring quinoid compounds may fulfill such a role, as evidenced by the enhancement of aerobic DCPIP reduction upon addition of catalytic amounts of caffeic and chlorogenic acid. The observation that PMS, caffeic and chlorogenic acid, biopterin, 6-hydroxy-2-amino-4-hydroxypteridine and a quinone extract of N. tabacum quenched the inhibitory effect of blue light on tobacco glycollate oxidase, is in accordance with the possible function of such compounds in glycollate oxidation.Abbreviation DCPIP 2,6-dichlorophenolindophenol - FMN flavin mononucleotide - PMS phenazine methosulphate     

An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium

An H₂O₂-requiring enzyme system was found in the extracellular medium of ligninolytic cultures of Phanerochaete chrysosporium. The enzyme system generated ethylene from 2-keto-4-thiomethyl butyric acid (KTBA), and oxidized a variety of lignin model compounds including the diarylpropane 1-(4′-ethoxy-3′-methoxyphenyl) 1,3-dihydroxy-2-(4″-methoxyphenyl)propane (I), a β-ether dimer 1-(4′-ethoxy-3′-methoxyphenyl)glycerol-β-guaiacyl ether (IV) and an olefin 1-(4′-ethoxy-3′-methoxy-phenyl)1,2-propene (VI). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures. In addition, the enzyme system partially degraded ¹⁴C-ring labeled lignin. The enzyme was not found in high nitrogen (N) cultures, nor in cultures of a ligninolytic mutant strain which is incapable of metabolizing lignin.