Biosynthesis of ethylene: Formation of ethylene from methional by a cell-free enzyme system from cauliflower florets

1. The formation of ethylene from cauliflower florets is stimulated by the addition of either methionine or its hydroxy analogue. 2. Formation of ethylene from these compounds may also be demonstrated in cell-free extracts, but the most rapid formation is achieved by the addition of methional. 3. Fractionation of such extracts has shown that both particulate and non-particulate fractions are necessary for the formation of ethylene from methionine or its hydroxy analogues, but only the non-particulate fraction is necessary for its formation from methional. 4. A study of this system has shown that the conversion of methional into ethylene requires the presence of two enzyme systems, the first generating peroxide and the second catalysing the conversion of methional into ethylene in the presence of peroxide. 5. The presence of a heat-stable factor in cauliflower extracts that is necessary for the full activity of the enzyme converting methional into ethylene has also been shown. 6. The nature of this factor is at the present unknown; it is not a metal nor is it identifiable with many of the known coenzymes.     

BIOGENESIS OF ETHYLENE

1. The main characteristics of the biosynthetic system forming ethylene in plant tissues have been reviewed. The dependence of synthesis on a liberal supply of oxygen is clearly indicated by the fact that atmospheres containing 3–5% oxygen prevent the synthesis in fruits. There is no close connexion between respiratory activity and synthesis. Ripening of fruits and the changes associated with it may be initiated by ethylene; under such conditions the progress of formation of the hydrocarbon is autocatalytic. 2. Synthesis appears to be dependent on some degree of cell organization, since it responds acutely to changes in toxcity, tissue wounding and tissue destruction. Homogenates of many plant tissues do not produce ethylene in vitro, and the inability to use such extracts has imposed serious restrictions on biochemical studies which have in the past been mainly concerned with tracer studies and the use of tissue slices. 3. The chief difficulty associated with tracer studies aimed at determining the nature of the precursor stems from the fact that the synthesis of ethylene is only a minor pathway on the general metabolism of the cell. Thus the ratio of CO₂ to ethylene production is of the order of 164 in the case of the apple and as high as 18,000 in the case of less vigorous producers of ethylene. The incorporation of label from labelled substrates which enter the general metabolism of the cell is thus usually very low, and this makes it difficult to determine whether the incorporation observed has any real physiological significance. In fact only where incorporation into ethylene relative to that into CO₂ is high, as is the case with methionine, can one conclude that the substance can be considered to be an immediate precursor. 4. Because of the difficulty of obtaining clear-cut results with tracer techniques, attention has been devoted to the production of ethylene by model systems from substances of physiological interest. The studies have revealed that many substances found in plant tissue can be decomposed to yield ethylene in model systems functioning under physiological conditions. Two such substances, which have received most attention, are methionine and linolenic acid, and conditions under which ethylene is formed from them have been described. 5. Such developments have stimulated research to obtain evidence for or against the operation of such model systems in vivo. Using tissue-slice techniques, methionine and linolenic acid have both been found to stimulate ethylene formation in tissue slices. 6. The first demonstration of the synthesis of ethylene in vitro by enzymes isolated from the florets of the cauliflower has now been reported. The system involves the intermediate formation of methional from methionine by enzymes contained in the mitochondria, and the subsequent enzymic decomposition of methional into ethylene by non-particulate enzymes. These latter consist of a glucose oxidase and a peroxidase. The glucose oxidase in the presence of its substrate generates hydrogen peroxide, and peroxidase, in the presence of two co-factors, ^-coumaric acid esters and methane sulphinic acid, utilizes the peroxide to produce ethylene from methional. Although all components of this system have been isolated from extracts of floret tissue, proof that this is the actual or only process in vivo for this or other plant tissue has not as yet been achieved. The more recent demonstration of the possible involvement of linolenic acid underlines the necessity for further work. 7. Whilst much work still remains to be done to establish the mechanism of synthesis, which may not be identical in different plants, the related question of the nature of the events which stimulate the tissue to produce ethylene remains to be answered. Recent work has suggested that these events, induced by ageing of the tissue, are associated with the synthesis of new enzyme proteins, which are themselves the cause of the rapid onset of synthesis of ethylene, observed in most fruits, at the climacteric. 8. Much more information on the nature of events leading to and changes associated with the ripening syndrome in fruits and onset of senescence in vegetable tissues is needed before authoritative answers can be given to any of the questions raised in this review.     

Inactivation of Firefly Luciferase and Rat Erythrocyte ATPase by Ultrasound

Previous work in our laboratories has shown that, amongst other effects, irradiation of frog skin with low intensity ultrasound causes reductions in the chemical driving force of the short-circuit current. This indicated that either the Na/K dependent A TPase or A TP availability were being reduced. We measured the effect of ultrasound irradiation on A TP and NA/K-dependent A TPase from inverted erythrocyte ghosts and on firefly luciferin and luciferase activity. Our findings demonstrate that ultrasonic cavitation-induced sonochemical reactions were responsible for irreversible inactivation of luciferase and ATPase but had little or no effect on ATP and luciferin. We measured the levels of hydrogen peroxide generated by ultrasound under the conditions of our experiments and found that it could account for only part of the enzyme inactivation observed. Free radical scavengers/antioxidants were capable of fully protecting the enzymes from ultrasound-induced inactivation. These findings demonstrate that, in addition to hydrogen peroxide, free radicals generated by ultrasound are responsible for the effects.     

Hydrogen peroxide produced by two amino acid oxidases mediates antibacterial actions

The antibacterial actions of two amino acid oxidases, a D-amino acid oxidase from hog kidney and a L-amino acid oxidase from the venom of Agkistrodon halys, were investigated, demonstrating that both enzymes were able to inhibit the growth of both Gram-positive and Gram-negative bacteria, and that hydrogen peroxide, a product of their enzymatic reactions, was the antibacterial factor. However, hydrogen peroxide generated in the enzymatic reactions was not sufficient to explain the degree to which bacterial growth was inhibited. A fluorescence labeling assay showed that both of these two enzymes could bind to the surfaces of bacteria. To the best of our knowledge, this is the first report regarding the antibacterial activity of the D-amino acid oxidases.     

Reactive oxygen and ethylene are involved in the regulation of regurgitant-induced responses in bean plants

Application of regurgitant from Leptinotarsa decemlineata Say on wound surfaces of one wounded leaf of intact bean (Phaseolus vulgaris L.) plants resulted in activation of ethylene biosynthesis followed by an increase of both peroxidase and polyphenol oxidase activity. The aim of the present investigation was to study the source of increased oxidative enzyme activities in regurgitant-treated bean leaves and to determine if hydrogen peroxide and ethylene biosynthesis is responsible for regurgitant-induced amplification of wound responses in bean plants. As the regurgitant contained relative high activities of both peroxidase and polyphenol oxidase, there is a possibility that increased enzyme activities in bean leaves following regurgitant treatment is an artifact of insect-derived enzymes. Localisation experiments and electrophoretic analysis revealed that only part of the increased enzyme activities could be attributed to regurgitant-derived enzymes. Both increase of ethylene production and oxidative enzyme activities depended on protein synthesis. To demonstrate if the increase of oxidative metabolism was ethylene-dependent, seedlings were pretreated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, and 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene action. Increase of both peroxidase and polyphenol oxidase activity in wounded and subsequently regurgitant-treated leaf was abolished by both aminooxyacetic acid and 1-MCP. Inhibitor studies indicated that H2O2 generated through NADPH oxidase and superoxide dismutase is necessary for regurgitant-induced increase of ethylene production and oxidative enzyme activities.     

Oxidation of methanol,formaldehyde and formate by catalase purified from methanol-grown Hansenula polymorpha

Catalase has been partially purified from cell-free extracts of methanol-grown Hansenula polymorpha and its peroxidative properties were studied. It was shown that the enzyme is capable of oxidizing methanol, formaldehyde and formate in the presence of hydrogen peroxide. The physiological significance of these reactions in the transduction of energy from the oxidation of methanol in yeasts is discussed.     

Augmentation of antioxidant enzymes in vascular endothelium

The endothelium is a key site of injury from reactive oxygen species that can potentially be protected by the antioxidant enzymes superoxide dismutase and catalase. Large proteins, such as superoxide dismutase and catalase, do not readily penetrate cell membranes, which limits their efficacy in protecting cells from cellular reactions involving both intracellularly and extracellularly generated reactive oxygen species. Two methods are described that promote enzyme delivery to cultured endothelial cells and confer increased resistance to oxidative stress. The first method is to entrap the antioxidant enzymes within liposomes, which then become incorporated by endothelial cells and can increase enzyme specific activities by as much as 44-fold within 2 h. The second method involves covalent conjugation of polyethylene glycol (PEG) to superoxide dismutase and catalase, a technique that increases circulatory half-life and reduces protein immunogenicity. Conjugation of PEG to superoxide dismutase and catalase increased cellular-specific activities of these enzymes in cultured endothelial cells (but at a slower rate than for liposome entrapped enzymes) and rendered these cells more resistant to oxidative stress. Both liposome-mediated delivery and PEG conjugation offer an additional benefit over native superoxide dismutase and catalase because they can increase cellular antioxidant activities in a manner that can provide protection from both intracellular and extracellular superoxide and hydrogen peroxide.     

Ethylene formation from methional

The biosynthetic precursor of ethylene is 3-methylthiopropanal (methional). It has been claimed that hydroxyl (HO·) radicals are involved in this biosynthetic sequence, and that the production of ethylene from methional can be used as a specific probe for the presence of the HO· radical. We have now shown, however, that a variety of organic radicals lead to the production of ethylene from methional. Clearly this reaction cannot be used to test for the presence of HO· radicals, and the mechanism for the conversion of methional to ethylene will have to be reexamined.     

Simultaneous Application of Glucose Oxidases and Peroxidases in Bleaching Processes

Peroxidases (POD) are used in textile decoloration and bleaching processes, but these enzymes are unfortunately inactivated rapidly at high hydrogen peroxide concentrations. A new concept has therefore been developed, which is based on a simultaneous application of glucose oxidase and peroxidase. Starting with glucose as a substrate for glucose oxidase (GOD), hydrogen peroxide was generated in situ. The freshly formed substrate H₂O₂ was immediately used by the POD oxidizing colored compounds in dyeing baths. For example, 20 mg of the dyestuff Sirius Supra Blue®FGG 200 % could be decolorized using 125 mg glucose which corresponds to 24 mg hydrogen peroxide. These experiments show that the enzyme cascade works in principle in homogeneous decoloration processes. The enzymes were not degraded by the oxidant, because under these conditions the stationary peroxide concentration is nearly zero over the whole reaction time. Moreover, experiments were carried out to check if this combined system with GOD, glucose and POD could be used even in heterogeneous systems such as the textile bleaching of natural cotton fibers. Starting from 55, a significant higher degree of whiteness (according to Berger) up to 66 could be obtained.     

An extracellular haem-protein from Coriolus versicolor

A haem-containing protein has been isolated from the growth medium of Coriolus versicolor, a wood-rotting basidiomycete. The polypeptide was identified as a ‘peroxidase-type’ haem protein of MW 53 700, which appeared to be a glycoprotein and had a protoporphyrin IX prosthetic group with a mid-point redox potential of ?121 mV. It also bound carbon monoxide suggesting it may act as an oxidase, and liberated hydroxyl radicals from hydrogen peroxide as measured by its ability to release ethylene from methional.     

Avoiding high-valent iron intermediates: superoxide reductase and rubrerythrin

The Fenton or Fenton-type reaction between aqueous ferrous ion and hydrogen peroxide generates a highly oxidizing species, most often formulated as hydroxyl radical or ferryl ([Fe(IV)O](2+)). Intracellular Fenton-type chemistry can be lethal if not controlled. Nature has, therefore, evolved enzymes to scavenge superoxide and hydrogen peroxide, the reduced dioxygen species that initiate intracellular Fenton-type chemistry. Two such enzymes found predominantly in air-sensitive bacteria and archaea, superoxide reductase (SOR) and rubrerythrin (Rbr), functioning as a peroxidase (hydrogen peroxide reductase), contain non-heme iron. The iron coordination spheres in these enzymes contain five or six protein ligands from His and Glu residues, and, in the case of SOR, a Cys residue. SOR contains a mononuclear active site that is designed to protonate and rapidly expel peroxide generated as a product of the enzymatic reaction. The ferrous SOR reacts adventitiously but relatively slowly (several seconds to a few minutes) with exogenous hydrogen peroxide, presumably in a Fenton-type reaction. The diferrous active site of Rbr reacts more rapidly with hydrogen peroxide but can divert Fenton-type reactions towards the two-electron reduction of hydrogen peroxide to water. Proximal aromatic residues may function as radical sinks for Fenton-generated oxidants. Fenton-initiated damage to these iron active sites may become apparent only under extremely oxidizing intracellular conditions.     

Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.     

Conjugation of enzymes on polymer nanoparticles covered with phosphorylcholine groups

We investigated the bioconjugation of enzymes on polymer nanoparticles covered with bioinert phosphorylcholine groups. A water-soluble amphiphilic phospholipid polymer (PMBN) was specially designed for preparation of nanoparticles and conjugation with enzymes on them. The PMBN was prepared by random copolymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate, and p-nitrophenylester bearing methacrylate. The PMBN was used as an emulsifier and a surface modifier to prepare the poly(l-lactic acid) nanoparticles by a solvent evaporation technique in aqueous medium. The nanoparticles covered with phosphorylcholine groups were stably dispersed in an aqueous solution and a phosphate buffered saline. The diameter and surface zeta-potential of the nanoparticles were ca. 200 nm and -6 mV, respectively. The p-nitrophenyl ester groups, which are active ester units for the amino groups of the protein, were located at the surface of the nanoparticles. Both acetylcholine esterase and choline oxidase were co-immobilized (dual-mode conjugation) by the reaction between the p-nitrophenyl ester group and the amino group of these enzymes. The enzymatic reactions on the nanoparticles were followed using a microdialysis biosensor system with a microtype hydrogen peroxide electrode in the probe. The nanoparticles conjugated with these enzymes could detect the acetylcholine chloride as hydrogen peroxide, which is a product of the enzymatic reactions on the surface of the nanoparticles in the probe. Namely, continuous enzyme reactions could be occurring on the surface of the nanoparticles. It is concluded that the nanoparticles are a promising tool for a highly sensitive and microdiagnostic system.     

Hydrogen peroxide-forming NADH oxidase belonging to the peroxiredoxin oxidoreductase family: existence and physiological role in bacteria

Amphibacillus xylanus and Sporolactobacillus inulinus NADH oxidases belonging to the peroxiredoxin oxidoreductase family show extremely high peroxide reductase activity for hydrogen peroxide and alkyl hydroperoxides in the presence of the small disulfide redox protein, AhpC (peroxiredoxin). In order to investigate the distribution of this enzyme system in bacteria, 15 bacterial strains were selected from typical aerobic, facultatively anaerobic, and anaerobic bacteria. AhpC-linked alkyl hydroperoxide reductase activities were detected in most of the tested strains, and especially high activities were shown in six bacterial species that grow well under aerobic conditions, including aerobic bacteria (Alcaligenes faecalis and Bacillus licheniformis) and facultatively anaerobic bacteria (Amphibacillus xylanus, Sporolactobacillus inulinus, Escherichia coli, and Salmonella enterica serovar Typhimurium). In the absence of AhpC, the purified enzymes from A. xylanus and S. inulinus catalyze the NADH-linked reduction of oxygen to hydrogen peroxide. Similar activities were observed in the cell extracts from each of these six strains. The cell extract of B. licheniformis revealed the highest AhpC-linked alkyl hydroperoxide reductase activity in the four strains, with V(max) values for hydrogen peroxide and alkyl hydroperoxides being similar to those for the enzymes from A. xylanus and S. inulinus. Southern blot analysis of the three strains probed with the A. xylanus peroxiredoxin reductase gene revealed single strong bands, which are presumably derived from the individual peroxiredoxin reductase genes. Single bands were also revealed in other strains which show high AhpC-linked reductase activities, suggesting that the NADH oxidases belonging to the peroxiredoxin oxidoreductase family are widely distributed and possibly play an important role both in the peroxide-scavenging systems and in an effective regeneration system for NAD in aerobically growing bacteria.     

Microbial metabolism of ethylene

The ethylene-oxidizing strain E20 was grown on different carbon sources to obtain information on the metabolism of ethylene from simultaneous adaptation studies and from measurements of specific activities of enzymes in cell-free extracts.From the simultaneous adaptation studies it was concluded that ethylene oxide is a product of ethylene catabolism. The bacterium was also able to grow on the epoxide. From a comparison of the specific activities of isocitrate lyase and malate synthetase in different extracts it was concluded that the glyoxylate cycle was involved in the metabolism of ethylene, indicating that acetyl-CoA is a metabolite of ethylene catabolism. The sequence of reactions leading from ethylene oxide to acetyl-CoA could not be established from the simultaneous adaptation experiments and the enzyme activities in extracts.Support for the research has come in part from grants of the N.V. Nederlandse Gasunie and the VEG Gasinstituut.     

Lactoperoxidase-catalyzed oxidation of [4-14C]estradiol

The conversion of [4-14C]estradiol to water-soluble products by lactoperoxidase (EC 1.11.1.7) in the presence of added or generated H2O2 was studied using albumin or tyrosine as acceptor. The enzyme was able to catalyze the oxidation and binding of estradiol to albumin even in the absence of 2,4-dichlorophenol at very low concentrations of hydrogen peroxide. Other systems in which H2O2 was replaced by oxygen and Mn2+, light-sensitized riboflavin or glutathione was also shown to be active in the conversion of estradiol to water-soluble products and the effect of inhibitors on these reactions was investigated. Possible mechanisms for the peroxidase-catalyzed formation of these estradio metabolites are discussed.     

Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack.

Reaction of certain peptides and proteins with singlet oxygen (generated by visible light in the presence of rose bengal dye) yields long-lived peptide and protein peroxides. Incubation of these peroxides with glyceraldehyde-3-phosphate dehydrogenase, in the absence of added metal ions, results in loss of enzymatic activity. Comparative studies with a range of peroxides have shown that this inhibition is concentration, peroxide, and time dependent, with H2O2 less efficient than some peptide peroxides. Enzyme inhibition correlates with loss of both the peroxide and enzyme thiol residues, with a stoichiometry of two thiols lost per peroxide consumed. Blocking the thiol residues prevents reaction with the peroxide. This stoichiometry, the lack of metal-ion dependence, and the absence of electron paramagnetic resonance (EPR)-detectable species, is consistent with a molecular (nonradical) reaction between the active-site thiol of the enzyme and the peroxide. A number of low-molecular-mass compounds including thiols and ascorbate, but not Trolox C, can prevent inhibition by removing the initial peroxide, or species derived from it. In contrast, glutathione reductase and lactate dehydrogenase are poorly inhibited by these peroxides in the absence of added Fe2+-EDTA. The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions. Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result in selective subsequent damage to other proteins, as evidenced by loss of enzymatic activity, via the formation and subsequent reactions of protein peroxides. These reactions may be important in the development of cellular dysfunction as a result of photo-oxidation.