Ascorbate Peroxide Research Articles

Metalliferous and non-metalliferous populations of Viola tricolor represent similar mode of antioxidative response

Heavy metal-contaminated sites are excellent areas to examine the antioxidative machinery responsible for physiological adaptations of many plant species. Superoxide dismutase (SOD), guaiacol peroxide (GPX), ascorbate peroxide (APX), catalase (CAT) activity and hydrogen peroxide (H(2)O(2)) content were analyzed in leaves and roots of Viola tricolor (Viola) from contaminated soils ('Bukowno', 'Saturn', 'Warpie' heaps), and non-contaminated soil ('Zakopane meadow') to examine the level of oxidative stress and antioxidative response. In leaves, six isoforms of SOD were recognized. Roots possessed two additional bands, named manganese superoxide dismutase (MnSOD)-like form (MnSODI) and Cu/ZnSOD-like form (Cu/ZnSODIV). The H(2)O(2) content in leaves ranged from 554 to 5 098 micromol H(2)O(2)/gf.w. and was negatively correlated with CAT activity. The non-contaminated population was characterized by the lowest CAT activity combined with the highest H(2)O(2) concentration. Two isoforms of CAT, CAT-1 and CAT-2, were recognized in leaves of plants from non-contaminated and contaminated sites, respectively. In roots of individuals from two heaps ('Warpie' and 'Saturn'), two distinct bands for each CAT isoform were observed. A slower migrating band may be an aggregate, exhibiting CAT and MnSODs activities. Both peroxidases (APX and GPX) presented the same pattern of activity, depending on the organ, indicating that in leaves and roots APX and GPX were regulated in parallel. Differences in enzyme activities and H(2)O(2) content between plants from different contaminated sites were statistically significant, but were tightly maintained at a very similar level. Prolonged and permanent heavy metal stress evoked a very similar mode of antioxidative response in specimens of analyzed metalliferous populations not causing measurable oxidative stress. Thus, our results clearly indicate that V. tricolor is a taxon well adapted to heavy metal-contaminated soils, and that differences in enzyme activities and H(2)O(2) content result from adjustment of plants to a variety of conditions.

Transcript levels of antioxidative genes and oxygen radical scavenging enzyme activities in chilled zucchini squash in response to superatmospheric oxygen

The transcript levels of antioxidative genes including Mn-superoxide dismutase (Mn-SOD), Cu/Zn SOD, ascorbate peroxidise (APX), and catalase (CAT) were relatively constant during storage at 5 °C with high oxygen treatment in freshly harvested zucchini squash ( Cucurbita pepo L. cv. Elite). However, the expressions of alternative oxidase (AOX) were induced slightly in squash treated with 60% and 100% oxygen for 3 days when compared with control squash. These increases in AOX transcript levels were correlated with the increased chilling resistance in the treated squash. The corresponding oxygen radical scavenging enzyme activities including SOD, APX, CAT, and peroxidise (POD) in treated samples were also higher than those in the control for the first 3 days at 5 °C. Transcript levels of AOX increased substantially between 3 and 6 days in all treatments suggesting the involvement of alternative respiratory pathway during chilling stress. All of the enzyme activities in 100% oxygen treated squash started to decline after 6 or 9 days of cold storage to a level comparable or lower than those of the control. These declines were correlated to the loss of chilling resistance in the 100% oxygen treated tissue as indicated in the chilling injury index. However, squash treated with 60% oxygen maintained elevated levels of all enzyme activities except POD and sustained the least chilling injury throughout the 15 days of storage at 5 °C. The oxygen radical absorbance capacity (ORAC) values and total phenol content remained high in squash treated with 60% and 100% oxygen for the first 9 days, then their levels in the 100% oxygen treated samples declined sharply while those in the 60% oxygen treated samples maintained elevated, indicating that both ORAC activity and phenolic content may also contribute to the resistance of tissue against chilling injury. The 100% oxygen treated squash showed the lowest respiration rate and 60% oxygen treated samples had the lowest ethylene production. These data may also be an indication of the low chilling injury in the high oxygen treated squash.

Variations in Growth, Photosynthesis and Defense System Among Four Weed Species Under Increased UV‐B Radiation

AbstractWeed tolerance of UV‐B radiation varies with species, and the radiation could affect weed ecology and management. Variations in growth, photosynthesis and defense system among four important agronomic weeds, Abutilon theophrasti Medik, Amaranthus retroflexus L., Digitaria sanguinalis (L.) Scop and Chloris virgata Swartz, under increased UV‐B radiation (ambient and increased radiation at 2.7, 5.4 and 10.8 kJ‐m−2‐d−1) were studied in the greenhouse experiment. After 2 weeks of radiation, the shoots’dry mass decreased with increasing UV‐B radiation except for D. sanguinalis. The reduction in biomass was the result of changes in morphology and physiology. Higher levels of UV‐B treatment decreased the leaf area, plant height, net photosynthetic rate and chlorophyll contents, while it increased the contents of wax and UV‐B absorbing compound in all species, except for A. retroflexus, which did not increase significantly. The activity of superoxide dismutase, catalase, ascorbate peroxide and the content of ascorbic acid changed differently among the weed species as UV‐B radiation increased. D. sanguinalis was the most tolerant and A. retroflexus the most sensitive to increased UV‐B radiation. The results also show that the two grass species (D. sanguinalis and C. virgata) were more tolerant to UV‐B radiation than the two broad‐leafed species (A. theophrasti and A. retroflexus). The UV‐B absorbing compound and leaf wax played important roles against UV‐B damages in the two grass weeds. The overall results suggest that weed community, competition and management will be altered by continuous ozone depletion.(Handling editor: Li‐Xin Zhang)

Damage of oxidative stress on mitochondria during microspores development in Honglian CMS line of rice

One of the cytoplasmic male sterility (CMS) types used for hybrid rice (Oryza sativa L.) production in China is the Honglian (HL)-CMS. Previous studies suggested that pollen abortion of the sterile plants was resulted from a special programmed cell death (PCD) program started at meiosis in the microspores. To elucidate the molecular basis of the pollen abortion, we compared the biochemical and physiological properties such as content of reactive oxygen species (ROS), ATP, NADH, total glutathione and ascorbate acid, the activities of dehydroascrbate reductase, glutathione reductase, ascorbate peroxides and superoxide dismutase, and the integrity of mitochondrial genome DNA isolated from an HL-CMS line, Yuetai A and its maintainer line, Yuetai B. Our results indicated that the mitochondria of the HL-CMS line suffered from a serious oxidative stress during microspores development. Oxidative stress induced by abnormal increased ROS at meiosis stage resulted in the depletion of ATP and NADH, and the degradation of mitochondrial genomic DNA. This suggests that the presence of redox signal originated in mitochondria affects the rest of the cell. Therefore, it is possible that the abortion of premature microspores in HL-CMS line is induced by the chronic oxidative stress in mitochondria in the early stage of pollen development.

Metabolic adaptations to mercury-induced oxidative stress in roots of Medicago sativa L.

Alfalfa ( Medicago sativa) roots were treated with mercuric ions in a concentration- and time-dependent manner, and lipid peroxidation was studied biochemically as well as histochemically along with other physiological responses. Histochemical staining with Schiff’s reagent and Evans blue revealed that the peroxidation of membrane lipids and loss of plasma membrane integrity in Hg-treated roots occurred in the meristem and the elongation zone. The histochemical observations were supported by the quantitative determinations of thiobarbituric acid reactive substances (TBARS). However, under the mercuric ions stress, the alfalfa plants showed no significant alteration of hydrogen peroxide in roots. Analysis of lipoxygenase activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that there were two isoforms in the root of alfalfa plants, but they showed quite different patterns under the Hg exposure. Also, using non-denaturing PAGE, activities of superoxide dismutase (SOD) and peroxidase (POD) were determined in roots after treatment with Hg ions. The total activities of SOD and POD increased in roots after Hg treatment of roots. Activity of ascorbate peroxides (APX) was stimulated at relatively high concentration of Hg (40 μM), and after prolonged Hg exposure (20 μM, 24 h). In contrast, glutathione reductase activity was depressed at higher concentrations of Hg (10–20 μM). Treatments of seedlings with 10–40 μM Hg decreased the ascorbate and glutathione amounts but increased total non-protein thiols. The above results indicated that Hg exerted its toxic effect on the root growth of alfalfa by induction of oxidative stress.

Effect Of Aqueous Extracts Of <I>Allium sativum</I> On Some Parameters Of Oxidative Stress In Mice Brain

This invitro study investigates the effects of Allium sativum L., Liliaceae, garlic on some markers of oxidative stress induced by hydroxyl radicals (OH - ) generated by Fenton's reaction and potentiated by L-glutamate. Garlic at doses between 50 200mg/mL significantly decreased the activities of catalase (CAT) and acetyl cholinesterase (AChE) in the brain tissues of experimental mice incubated with iron-hydrogen peroxide ascorbate in the absence (CAT, 3.1 ± 0.9 IU/L; AChE, 0.276 ± 0.05 ΔOD/min) or presence (CAT, 1.85 ± 0.8 IU/L; AChE, 0.135 ± 0.04 ΔOD/min) of 50mM L-glutamate (Control values: 12.5 ± 0.8 IU/L and 0.576 ± 0.09 OD/min; P Allium sativum possessed neuroprotective and anti-DNA damaging effects. Key words: Allium sativum L., Liliaceae, garlic, oxidative stress, catalase, acetyl cholinesterase, brain tissues, mice. Nig. J. Nat. Prod. and Med. Vol.8 2004: 13-18

Radical induced damage ofMicrococcus luteusbacteria monitored using FT‒IR spectroscopy

Oxidative damage induced by ascorbic acid (AA) and hydrogen peroxide (H2O2) was monitored by Fourier transform infrared spectroscopy (FT‒IR); it appeared as a rapid and convenient means to follow the biochemical changes generated in the culture media of the yellow-pigmentedMicrococcus luteus. Beyond a threshold of 20 mM for AA and of 40 mM for H2O2(final concentration), antioxidant systems were overwhelmed and significant changes were observed in the bacterial spectra, particularly in the 1430–900 cm−1region; this spectral window provided large information about carboxylate groups, phosphate-carrying compounds and polysaccharides implicated in the radical process. The spectroscopic results indicated that for the same final concentration, the toxicity of H2O2was less important than that of AA towardM. luteuscells, although H2O2had a more damaging effect on proteins. Thus, FT-IR spectroscopy was an appropriate physico-chemical tool suitable in biochemical and clinical research for early characterization of any type of radical aggression, and for rapid detection of the damage intensity.

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H2O2 concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H2O2 concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H2O2, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H2O2 and ABA-induced activities of APX and GR. ABA-induced accumulation of H2O2 was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H2O2 is involved in ABA-induced APX and GR activities in rice roots.

Peptide damage under Fenton conditions is sequence-dependent.

Peptides with two or more acidic amino acids are damaged to a greater extent than other peptides under Fenton conditions as revealed by treating a 29791 membered one-bead-one-compound peptide library with FeCl(3), sodium ascorbate and hydrogen peroxide.

A comparison of mechanisms of desiccation tolerance among three angiosperm resurrection plant species

The mechanisms of protection against mechanical and oxidative stress were identified and compared in the angiosperm resurrection plants Craterostigma wilmsii, Myrothamnus flabellifoliusand Xerophyta humilis. Dryinginduced ultrastructural changes within mesophyll cells were followed to gain an understanding of the mechanisms of mechanical stabilisation. In all three species, water filled vacuoles present in hydrated cells were replaced by several smaller vacuoles filled with non-aqueous substances. In X. humilis, these occupied a large proportion of the cytoplasm, preventing plasmalemma withdrawal and cell wall collapse. In C. wilmsii, vacuoles were small but extensive cell wall folding occurred to prevent plasmalemma withdrawal. In M. flabellifolius ,s ome degree of vacuolation and wall folding occurred, but neither were sufficient to prevent plasmalemma withdrawal. This membrane was not ruptured, possibly due to membrane repair at plasmodesmata junctions where tearing might have occurred. In addition, the extra-cytoplasmic compartment appeared to contain material (possibly similar to that in vacuoles) which could facilitate stabilisation of dry cells. Photosynthesis and respiration are particularly susceptible to oxidative stress during drying. Photosynthesis ceased at high water contents and it is proposed that a controlled shut down of this metabolism occurred in order to minimise the potential for photo-oxidation. The mechanisms whereby this was achieved varied among the species. In X. humilis, chlorophyll was degraded and thylakoid membranes dismantled during drying. In both C. wilmsii and M. flabellifolius, chlorophyll was retained, but photosynthesis was stopped due to chlorophyll shading from leaf folding and anthocyanin accumulation. Furthermore, in M. flabellifolius thylakoid membranes became unstacked during drying. All species continued respiration during drying to 10% relative water content, which is proposed to be necessary for energy to establish protection mechanisms. Activity of antioxidant enzymes increased during drying and remained high at low water contents in all species, ameliorating free radical damage from both photosynthesis and respiration. The nature and extent of antioxidant upregulation varied among the species. In C. wilmsii, only ascorbate peroxidise activity increased, but in M. flabellifolius and X. humilis ascorbate peroxidise, glutathione reductase and superoxide dismutase activity increased, to various extents, during drying. Anthocyanins accumulated in all species but this was more extensive in the homoiochlorophyllous types, possibly for protection against photo-oxidation.

ROLE OF IRON IN ASBESTOS-BODY-INDUCED OXIDANT RADICAL GENERATION

Asbestos bodies (AB) were harvested from human lung tissue digests and isolated from uncoated asbestos fibers. Samples containing 1000 AB were added to a reactive solution to investigate the ability of AB to oxidize deoxy-D-ribose and generate reactive oxygen species (ROS) in the presence of ascorbate and hydrogen peroxide as determined by formation of thiobarbituric acid (TBA)-reactive products. Three types of asbestos fibers were tested for comparison, since they are known to be able to produce ROS. The absorbance values measured with 1000 AB were significantly higher than those observed with 1000 fibers of the three types of asbestos. Since in our reaction system the only source of transition metals was the iron-rich AB, data suggest iron derived from the ferritin coating of AB was involved in oxidant generation. Addition of iron to AB enhanced TBA-reactive product formation, while chelation of Fe with deferoxamine reduced this reaction. Hydroxyl radical scavengers 1,3-dimethyl-2-thiourea (DMTU) and mannitol (MN) also effectively blocked TBA-reactive product generation. Data indicate the importance of Fe in ABinduced oxidant damage. With the addition of polymorphonuclear leukocytes (PMN) to AB, incubation in the reactive solution gave very high amounts of TBA-reactive products, but using a reactive solution devoid of ascorbate, very low amounts of TBA-reactive products were generated. In the latter condition, the superoxide of cell membranes probably reduced and removed iron from AB-coating ferritin, but less effectively than ascorbate. Further after the possible reoxidation of Fe2+, Fe3+ could be coordinated by lactoferrin. Since such availability of reductant is never approached in living systems, the iron in the AB coating is unlikely to function as a catalyst of Fenton-type reactions in vivo. damage to DNA. However, under in vivo conditions, and macrophages are associated with AB. The leukocyte membranes contain lactoferrin, which is an iron chelator provide protection against radical-induced cellular damage al., 1988; Afeltra et al., 1997). of this study was (1) to determine whether AB in vitro released with consequent hydroxyl radical generation and toxicity and

Visualizing tertiary folding of RNA and RNA-protein interactions by a tethered iron chelate: analysis ofHIV-1 Tat-TAR complex.

Replication of human immunodeficiency virus type 1 (HIV-1) requires specific interactions of Tat protein with the trans -activation responsive region (TAR) RNA, a 59 base stem-loop structure located at the 5'-end of all HIV transcripts. We have used an intramolecular RNA self-cleaving strategy to determine the folding of TAR RNA and its interactions with a Tat peptide. We incor-porated an EDTA analog at position 24 in the HIV-1 Tat binding site of the TAR RNA. After isolation and purification of the EDTA-TAR conjugate, RNA self-cleavage was initiated by the addition of an iron salt, ascorbate and hydrogen peroxide. Hydroxyl radicals generated from the tethered Fe(II) cleaved TAR RNA backbone in two localized regions. Sites of RNA cleavage were mapped by sequencing reactions. A Tat fragment, Tat(38-72), specifically inhibited RNA self-cleavage. To determine the structural changes caused by the Tat peptide, we performed Fe(II)-EDTA footprinting experiments on Tat-TAR complex. Our high-resolution footprinting results suggest that the inhibition of self-cleavage of EDTA-TAR is due to two effects of Tat binding: (i) Tat binds in the bulge and protects residues in the vicinity of the bulge from self-cleavage and (ii) RNA goes through a structural change where EDTA-U24 is rigidly positioned out of the helix and cannot get access to other nucleotides in the loop of TAR RNA, which are not protected by the Tat peptide. Our results demonstrate that Fe(II)-EDTA-mediated RNA self-cleavage can be applied to study RNA tertiary structures and RNA-protein interactions.

Preparation and properties of polybenzidine film-coated electrode as an H2O2 selective polymeric material

By means of electrochemical polymerization, polybenzidine-modified electrodes were prepared in an aqueous monomer solution at a potential of 0.7 V versus Ag/AgCl. The permselective character of the polybenzidine electrode prepared in a one-step procedure was examined for electroactive (ascorbic acid, oxalic acid, and hydrogen peroxide) and nonelectroactive (lactose, sucrose, and urea) species. Influence of the various parameters on the permselective properties of the polybenzidine membrane was systematically investigated and the optimal values for these parameters were determined. It has been found that polybenzidine membrane showed selective permeation for hydrogen peroxide while blocking the permeation of electroactive and nonelectroactive interferents through film. In brief, it is claimed that this polybenzidine film can be used as a coating material to prevent interferences in electrochemical biosensor applications. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 70: 2227–2234, 1998

Synthesis and optimization of permselective polymer (polyindoline) film

A polyindoline permselective polymer film was readily synthesized by an electrochemical polymerization of indoline in an aqueous solution of KCl at a potential of 0.4 V vs Ag/AgCl. The amperometric responses of the polyindoline film-coated platinum electrodes to electroactive (ascorbic acid, oxalic acid and hydrogen peroxide) and non-electroactive (lactose, sucrose and urea) substances were measured at a potential of 0.7 V. Effects of various variables such as film thickness, concentrations of monomer and electrolyte, and pH on the permselective behavior of the polymeric membrane were systematically investigated and the optimal values were determined. It was found that permselective polyindoline-coated electrodes prepared in this one-step procedure permitted hydrogen peroxide oxidation while it prevented interference due to oxidizable species such as ascorbic acid and oxalic acid. As a result, it is believed that this polymeric membrane, owing to its permselective character, can be used as the protective material in the construction of hydrogen peroxide-based biosensors.

Continuous detection of radiation or metal generated hydroxyl radicals within core chromatin particles

Purpose: The aim of this work was to adapt a recently developed fluorometric method for use in the detection of hydroxyl radical (HO) generated in the immediate vicinity of chromatin core particles reconstituted from pUC19 plasmid DNA and isolated core histones. Materials and methods: The procedure followed involves labelling nucleosomal histones with SECCA, a non-fluorescent coumarin derivative that generates the fluorescent 7-hydroxy-SECCA after reaction with HO. Core particles are formed using histones and pUC19 DNA in a salt-dialysis procedure. Results: Electron microscopy and micrococcal nuclease digestion are consistent with successful formation of core particles. No significant differences between core particle formation in the unlabelled and SECCA-labelled samples were detected. Exposure to HO generated by radiation or copper-- ascorbic acid-hydrogen peroxide results in a gradual induction of fluorescence. Studies using dimethyl sulphoxide (DMSO) demonstrate that, unlike HO produced by radiation, the majority of HO generated by copper-- ascorbic acid--hydrogen peroxide occurs primarily in the immediate vicinity of core particles and DNA and cannot be scavenged. Conclusions: The present procedure demonstrates the feasibility to quantitate HO generated by several agents in the immediate vicinity of nucleosomes (chromatin-associated HO) or associated with specific regions within the genome.

QUANTITATION OF PROTEIN DAMAGE IN METAL ION-CATALYZED OXIDATION SYSTEMS

The loss of enzymatic activity of lactate dehydrogenase was studied in several ascorbate, iron and hydrogen peroxide metal catalyzed oxidation solutions in which the initial concentration of each reactant was varied independently. Nonmonotonic concentration dependencies of enzymatic inactivation were observed for all three reactants. with minimum activity levels occurring in the 0·1 to 8 mM range. A first effort has been made to predict these concentration dependencies with a mathematical simulation model. The model consisted of the most commonly reported reactions and assumed that protein damage occurred through reaction with the hydroxyl radical. The simulation predicted nonmonotonic concentration dependencies of enzyme inactivation on each of the reactants. The predicted concentrations of the minima differed from the experimentally observed points by a factor of 2 to 4. Mathematically the minima occurred because each reactant was reported to react with hydroxyl radicals and form less reactive compounds. The plausibility that competition caused or contributed to the extrema was further explored with experimental competition studies between peroxide and the radical scavenger dimethylsulfoxide. A point of maximum hydroxyl radical formation was observed with increasing peroxide concentration. This maxima corresponded to the point of maximum lactate dehydrogenase damage observed with increasing peroxide concentration.

Antibacterial activity of copper(II) and cobalt(II) complexes with 1-(5-nitrofurfurylideneamino)-1,3,4-triazole in the presence of sodium ascorbate and hydrogen peroxide

Antibacterial activity of copper(II) and cobalt(II) complexes with 1-(5-nitrofurfurylideneamino)-1,3,4-triazole in the presence of sodium ascorbate and hydrogen peroxide

Lipid peroxidation inhibits acyl-CoA :- 1-acyl- sn-Glycero-3-phosphocholine O-acyltransferase but not CTP: Phosphocholine cytidylyltransferase activity in rat brain membranes

In brain tissue in vivo peroxidized according to three model systems, we determined two microsomal enzyme activities involved in phospholipid biosynthesis. The first, short-term model, was based on the i.v. administration to normal rats, twice a day, for a period of 1 week, of a sonicated emulsion of a peroxidized mixture of phospholipids and linoleate (4:1, w/w; 500 mg/day; hydroperoxides: 200–250 nmol/mg lipid). The half-life time of the injected toxic lipid species in the blood circulation was about 1 h. At the end of the week's treatment, brain and liver malondialdehyde, conjugated diene and lipid hydroperoxide levels were significantly higher in treated rats than in the controls. The second model consisted of the acute injection of aqueous Fe 2+ solution (50 mM) into lateral ventricles, and the collection of brain tissue 2 h later. The third model was based on two consecutive injections of hydroperoxylinoleate (1 mg each) into lateral ventricles over a period of 18 h, and the collection of brain tissue 2 h after the second administration. In brain microsomal membranes prepared from peroxide- or iron-treated rats, lysophosphatidylcholine acyltransferase activity exhibited a significant inhibition. On the contrary, in microsomal preparations derived from the short-term model, CTP : phosphocholine cytidylyltransferase activity was slightly stimulated. Intraventricular injection of linoleate or linoleic acid hydroperoxide left this enzyme activity unchanged. The effect of in vitro membrane peroxidation on both microsomal enzyme activities was investigated. By using an Fe 2+ (20 μM)-ascorbate (0.25 mM) peroxidation system, the residual acyltransferase and cytidylyltransferase activities were 80 and 72% of initial activity respectively. Significant dose-dependent inactivation of acyltransferase (maximum loss of 45% of initial activity) was seen when 0.1–10 μmol of photooxidized phospholipids were preincubated with 100 μg of microsomal membranes. Unoxidized or photooxidized phospholipids (1 mM) promoted a slight stimulation of cytidylyltransferase activity. Altogether, the results suggest a link between oxygen radical generation and the perturbation of the membrane structure in which the enzymes are located.

Lipid peroxidation inhibits oleoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine O-acyltransferase in rat CNS axolemma-enriched fractions.

The effect of phospholipid peroxidation on the acylation of lysoPtdCho (lysophosphatidylcholine) by axolemma-enriched fraction prepared from rat brain stem was investigated. After two types of peroxidative treatments, the in vitro induction of malondialdehyde and conjugated dienes formation in axolemmal membranes correlated to a shift in the ratio of saturated/unsaturated fatty acids. By using an Fe2+ (20 microM)-ascorbate (0.25 mM) peroxidation system, the residual acyltransferase activity was 55% of the initial one. No change in Km value for either oleoyl-CoA or lysoPtdCho was found, whereas a loss of 24% in Vmax was observed. After 5 min preincubation with 150 mM t-BuOOH, 70% inactivation of the acylation reaction was observed. A near suppression of enzyme activity was reached with 400 mM. The apparent Km for oleoyl-CoA decreased sharply (from 6.6 microM in control preparations to 4.1 microM in t-BuOOH-treated membranes), indicating a 2-fold increase in the enzymatic affinity for this substrate. The apparent Km for lysoPtdCho increased markedly (from 1.56 microM in the control preparations to 5.88 microM in t-BuOOH-treated membranes) whereas a decrease of Vmax (from 1.65 to 0.80 nmol/min/mg protein) for the same substrate was observed. Significant enzyme inactivation (loss of 60% of initial activity) was seen when 10 mumol of photooxidized phospholipids were preincubated with axolemmal membranes. Significant dose-dependent enzyme inactivation was brought about by addition of 10-60 mumol of peroxidized PtdEtn/100 micrograms axolemmal protein. The percent enzyme inhibition by peroxidized PtdCho at equivalent amounts was lower than that by PtdEtn.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydroxyl radical scavenging activity of compatible solutes

Compatible solutes were assessed for their hydroxyl radical scavenging activity by their ability to compete in two different hydroxyl radical generating and detecting systems. Hydroxyl radicals were generated by ascorbate-hydrogen peroxide or by xanthine oxidase-hypoxanthine-hydrogen peroxide. They were detected by hydroxylation of salicylate or by denaturation of malate dehydrogenase. Of the compatible solutes tested, sorbitol, mannitol, myo-inositol and proline were effective hydroxyl radical scavengers. Glycinebetaine was ineffective. The role of compatible solutes as hydroxyl radical scavengers in vivo is discussed.