Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid. Implications for antioxidant protection in the inflamed rheumatoid joint

Ascorbic acid, at physiological concentrations, can scavenge the myeloperoxidase-derived oxidant hypochlorous acid at rates sufficient to protect alpha 1-antiprotease against inactivation by this molecule. The rapid depletion of ascorbic acid at sites of inflammation, as in the inflamed rheumatoid joint, may therefore facilitate proteolytic damage.     

Evaluation of the Antioxidant Actions of Ferulic Acid and Catechins

We have evaluated the abilities of ferulic acid, (±) catechin, (+) catechin and (-) epicatechin to scavenge the reactive oxygen species hydroxyl radical (OH±), hypochlorous acid (HOCl) and peroxyl radicals (RO₂).

Ferulic acid tested at concentrations up to 5 mM inhibited the peroxidation of phospholipid liposomes. Both (±) and (+) catechin and (-) epicatechin were much more effective. All the compounds tested reacted with trichloromethyl peroxyl radical (CCl₃O₂) with rate constants > 1 × 10⁶M^?1s^?1.

A mixture of FeCl₃-EDTA, hydrogen peroxide (H₂O₂) and ascorbic acid at pH 7.4, has often been used to generate hydroxyl radicals (OH.) which are detected by their ability to cause damage to the sugar deoxyribose. Ferulic acid, (+) and (±) catechin and (-) epicatechin inhibited deoxyribose damage by reacting with OH. with rate constants of 4.5 × 10⁹M^?1s^?1, 3.65 × 10⁹M^?1s^?1, 2.36 × 10⁹M^?1s^?1 and 2.84 × 10⁹M^?1s^?1 respectively. (-) Epicatechin, ferulic acid and the (+) and (±) catechins exerted pro-oxidant action, accelerating damage to DNA in the presence of a bleomycin-iron complex. On a molar basis, ferulic acid was less effective in causing damage to DNA compared with the catechins.

A mixture of hypoxanthine and xanthine oxidase generates O₂ which reduces cytochrome c to ferrocytochrome c. (+) Catechin and (-) epicatechin inhibited the reduction of cytochrome c in a concentration dependent manner. Ferulic acid and (±) catechin had only weak effects.

All the compounds tested were able to scavenge hypochlorous acid at a rate sufficient to protect alpha-1-antiproteinase against inactivation. Our results show that catechins and ferulic acid possess antioxidant properties. This may become important given the current search for “natural” replacements for synthetic antioxidant food additives.     

Interaction of tert -butyl Hydroperoxide with Hypochlorous Acid. A Spin Trapping and Chemiluminescence Study

The formation of radical species during the reaction of tert-butyl hydroperoxide and hypochlorous acid has been investigated by spin trapping and chemiluminescence. A superposition of two signals appeared incubating tert-butyl hydroperoxide with hypochlorous acid in the presence of the spin trap &#102 -(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). The first signal (a_N = 1.537mT, a^&#103_H = 0.148mT) was an oxidation product of POBN caused by the action of hypochlorous acid. The second spin adduct (a_N = 1.484mT, a^&#103_H = 0.233mT) was derived from a radical species that was formed in the result of reaction of tert-butyl hydroperoxide with hypochlorous acid. Similarly, a superposition of two signals was also obtained using the spin trap N-tert-butyl- &#102 -phenylnitrone (PBN). tert-Butyl hydroperoxide was also treated with Fe²⁺ or Ce⁴⁺ in the presence of POBN. Using Fe²⁺ a spin adduct with a _N= 1.633mT and a^&#103_H = 0.276mT was observed. The major spin adduct formed with Ce⁴⁺ was characterised by α_N = 1.480mT and a^&#103_H = 0.233mT. The reaction of tert-butyl hydroperoxide with hypochlorous acid was accompanied by a light emission, that time profile and intensity were identical to those emission using Ce⁴⁺. The addition of Fe²⁺ to tert-butyl hydroperoxide yielded a much smaller chemiluminescence. Thus, tert-butyl hydroperoxide yielded in its reaction with hypochlorous acid or Ce⁴⁺ the same spin adduct and the same luminescence profile. Because Ce⁴⁺ is known to oxidise organic hydroperoxides to peroxyl radical species, it can be concluded that a similar reaction takes place in the case of hypochlorous acid.     

Acid Protease of Bovine Milk

Occurrence of milk acid protease in bovine casein in addition to alkaline protease was found and purification of this enzyme was achieved. The enzyme had a pH optimum at 4.0 and was most stable at pH 3.5. The molecular weight of the enzyme was 36,000 and no inhibition was observed by diisopropyl-fluorophosphate, EDTA etc. This enzyme is considered to be similar to cathepsin D.

Milk acid protease mainly hydrolyzed α_s-casein and similar change was observed in autolysis of casein at pH 5.5. It is suggested that milk acid protease may have some significance in cheese ripening.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Ricinoleic Acid Catabolism in Peroxisomes

Peroxisomes from castor bean endosperm and mung bean hypocotyl completely degrade ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) to acetyl-CoA. Concomitant NADH formation occurred with a stoichiometry of 9 nmol NADH formed per 1 nmol ricinoleate degraded. At the C₈-intermediate level, where the hydroxy group of ricinoleic acid forms a barrier to β-oxidation, 2-hydroxyoctanoate and 2-oxooctanoate were detected as intermediates. 2-Hydroxyoctanoate was oxidized to 2-oxooctanoate with H₂O₂ producing a reaction exhibiting 1:1 stoichiometry of the products. The peroxisomes appeared to oxidize both isomers of racemic 2-hydroxyoctanoate. 2-Oxooctanoate was metabolized to heptanoyl-CoA (propionyl-CoA and acetyl-CoA) in a NAD-dependent, but ATP-independent, reaction. Heptanoate was not detected as an intermediate. Imidazole, an inhibitor of α-oxidation, did not effect the degradation of ricinoleate or 2-oxooctanoate. Arsenite, an inhibitor of oxidative decarboxylation, inhibited the metabolism of ricinoleate at the C₈-intermediate level, according to the accumulation of 2-oxooctanoate and the stoichiometry of concomitant NADH formation. Arsenite completely inhibited the metabolism of 2-oxooctanoate. It is concluded that the barrier caused by the hydroxy group of ricinoleic acid and prevention of β-oxidation at the C₈-intermediate level, is circumvented by an α-hydroxy acid oxidase reaction followed by an oxidative decarboxylation allowing return to the β-oxidation track.     

The Biological and Structural Similarity between Lunularic Acid and Abscisic Acid

Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA₃)-induced α-amylase induction in embryoless barley seeds at 120 μM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 μM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C₁-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.     

Metabolism of Chiral Ionylideneacetic Acids on the Abscisic Acid Biosynthetic Pathway in Cercospora

A Chiralcel OJ column was used to determine the absolute configuration of naturally occurring α-ionylideneacetic acid from Cercospora rosicola and γ-ionylideneacetic acid from C. cruenta as (R) enantiomers in accordance with their biosynthetic product, (S)-ABA. Both enantiomers of [1, 2-¹³C₂]-α and γ-ionylideneacetic acids were prepared and fed to C. rosicola and C. cruenta. Six combinations of feeding experiments comparatively and unequivocally demonstrated stereoselectivity in the biosynthetic conversions, including stepwise hydroxylation at C-1′ and 4′. Enzymatic isomerization from the γ to α-intermediate was suggested not to be involved in ABA biosynthesis in C. rosicola.     

Acid tolerance mediated by membrane ATPases in Lactobacillus acidophilus

The acid tolerance response in Lactobacillus acidophilus CRL 639, induced at pH 4.2 for 15 min, is mediated by the cell membrane F ₁-F ₀ ATPase. The specific activity of the enzyme was induced 1.6-fold after acid adaptation compared to a non-adapted control. The ATPase was optimal at pH 6 with a K _m=0.8 mM and a V _max=100 mM.     

Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Twelve acid hydrolases, 4 near-neutral hydrolases and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase had a broad pH-activity curve from 4.0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

模拟秋季酸雨对三角枫叶片光合生理特性的影响

以三角枫幼苗为材料,采用盆栽方法,研究了pH 5.6、pH 4.0、pH 3.0和pH 2.0酸度模拟酸雨胁迫对三角枫叶片光合生理特性的影响,以探讨酸雨胁迫下三角枫的光合生理响应机制。结果显示:(1)随着酸雨浓度的增加和胁迫时间的延长,三角枫叶片相对叶绿素含量下降幅度逐步增大;叶片丙二醛含量呈逐渐上升的趋势,且各处理均显著高于对照;叶片质膜透性和脯氨酸含量均呈先升高后降低的趋势,且pH 2.0处理的叶片质膜透性在试验20d时迅速增大,升高幅度最大(146.3%)。(2)叶片净光合速率、蒸腾速率、水分利用效率、表观光能利用效率以及表观CO2利用效率在胁迫下也均呈显著下降趋势,且以pH 3.0和pH 2.0处理降幅最大。研究表明,pH 4.0的模拟酸雨对三角枫叶片的光合生理指标无显著影响,而pH≤3.0的强酸雨胁迫使三角枫叶片叶绿素含量显著下降、膜保护系统受损、光合作用效率显著下降;三角枫能适应弱酸雨(pH≥4.0)环境,可作为酸雨地区的园林绿化树种。     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

Acid carboxypeptidase from a wood-deteriorating basidiomycete, Pycnoporus Sanguineus

An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a K_m of 0.74 mM and a k_cat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The K_m and k_cat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

Abstract

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl^-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1×10⁷ M^-1s^-1 (pH 5), 2.0×10⁸ M^-1s^-1 (pH 7) and 2.0×10⁶ M^-1s^-1 (pH 9) at 15°C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H₂O₂, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c_50%=258±24 μM HOCl) and bimane-S-glutathione (B-SG, c_50%=125±16 μM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH]_oxidized=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg²⁺-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c_50%=478±36 μM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.     

Purification and Characterization of an Enzyme That Catalyzes Ring Cleavage of Aspergillic Acid,from Tvichoderma koningii ATCC 76666

The aspergillic acid degrading enzyme (ADE) that catalyzes the cleavage of the pyrazine ring in aspergillic acid (AA, l-hydroxy-3-isobutyl-6-sec-butyl-2-pyrazinone) was purified to electrophoretic homogeneity from extracts of Trichoderma koningii ATCC 76666. ADE was a homodimeric protein with a molecular mass of 112kDa, contained lmol of FAD per mol of subunit, and required NAD(P)H and molecular oxygen for its activity. ADE had an isoelectric point of around 5.3, and an optimum pH of 7.0–8.0. p-Chloromercuribenzoate and HgCl₂ completely inhibited ADE activity, while metal chelating reagents, α, α′-dipyridyl and o-phenanthroline, were not inhibitors. The substrate specificity among AA-related compounds was that hydroxyaspergillic acid was a poor substrate (16% of the activity for AA) and deoxyaspergillic acid did not serve as a substrate.     

The free amino acid tyrosine enhances the chlorinating activity of human myeloperoxidase

A key function of neutrophil myeloperoxidase (MPO) is the synthesis of hypochlorous acid (HOCl), a potent oxidizing agent that plays a cytotoxic role against invading bacteria and viruses at inflammatory sites and in phagosomes. MPO displayed a chlorinating activity preferably at acidic pH but at neutral pH MPO catalyzes mainly reactions of the peroxidase cycle. In the present work effects of tyrosine on the chlorinating activity of MPO were studied. At pH 7.4 we detected an increased HOCl production in the presence of tyrosine not only by the MPO-H₂O₂-Cl^- system but also in suspensions of zymosan-activated neutrophils. An excess of H₂O₂ is known to cause an accumulation of compound II of MPO blocking the generation of HOCl at neutral pH. As evidenced by spectral changes, tyrosine-induced activation of MPO to synthesize HOCl was due to the ability of tyrosine to reduce compound II back to the native state, thus accelerating the enzyme turnover. MPO-induced oxidation of tyrosine is relevant to what can be in vivo; we detected MPO-catalyzed formation of dityrosine in the presence of plasma under experimental conditions when tyrosine concentration was about three magnitudes of order less than the Cl⁻ concentration. At acidic pH formation of compound II was impaired in the presence of chloride and dityrosine couldn't be detected in plasma. In conclusion, the ability of tyrosine to increase the chlorinating activity of MPO at neutral pH and enhanced values of H₂O₂ may be very effective for the specific enhancement of HOCl production under acute inflammation.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Bromination and chlorination reactions of myeloperoxidase at physiological concentrations of bromide and chloride

Myeloperoxidase and eosinophil peroxidase use hydrogen peroxide to oxidize halides and thiocyanate to their respective hypohalous acids. Myeloperoxidase produces mainly hypochlorous acid and hypothiocyanite. Hypobromous acid and hypothiocyanite are the major products of eosinophil peroxidase. We have investigated the ability of myeloperoxidase to produce hypobromous acid in the presence of physiological concentrations of chloride and bromide. In accord with previous studies, between pH 5 and 7, myeloperoxidase converted about 90% of available hydrogen peroxide to hypochlorous acid and the remainder to hypobromous acid. Above pH 7, there was an abrupt rise in the yield of hypobromous acid. At pH 7.8, it accounted for 40% of the hydrogen peroxide. Bromide, at physiological concentrations, promoted a dramatic increase in bromination of human serum albumin catalyzed by myeloperoxidase. The level of 3-bromotyrosine increased to 16-fold greater than that for 3-chlorotyrosine. Chlorination of tyrosyl residues was not affected by bromide. With reagent hypohalous acids, bromination of tyrosyl residues was considerably more facile than chlorination. Hypochlorous acid promoted bromination to only a limited extent, which ruled out transhalogenation as a substantive route to 3-bromotyrosine. Chloramines and bromamines were also formed on albumin. Bromamines decayed much faster than chloramines and rapidly gave rise to protein carbonyls. We conclude that at physiological concentrations of chloride and bromide, hypobromous acid can be a major oxidant produced by myeloperoxidase. Its production in vivo will depend on pH and the concentration of bromide. Once produced, hypobromous acid will react with proteins to form bromamines, carbonyls, and brominated tyrosine residues. Consequently, 3-bromotyrosine should be considered as an oxidative product of myeloperoxidase and cannot be used as a specific biomarker for eosinophil peroxidase.     

Amino Acid Composition of Cucumber Green Mottle Mosaic Virus Coat Protein and Sequence Determination of Its Tryptic Peptides

Amino acid composition of the CGMMV* coat protein was determined to be as follows: Asp₂₀, Thr₁₀, Ser₂₄, Glu₁₀. Pro₆, Gly₉, Ala₂₁, Val₇, Ile₇, Leu₁₈, Tyr₄, Phe₉, Lys₄, His₁, Arg₈, Trp₂. No terminal α-amino group was detected by dinitrophenylation method. The carboxyl-terminus was found to be serine by hydrazinolysis of the protein and digestion with carboxypeptidase A.

For sequence analysis of the coat protein, tryptic digestion was accomplished at pH 8.0 resulting in ten soluble and several insoluble peptides at pH 4.5. The amino acids contained in soluble peptides accounted for 91 out of 160 residues in the whole protein. The amino acid sequences of ten soluble peptides were determined.

From the similarities of amino acid sequence of the peptides to those of TMV* protein, CGMMV was assumed to be a strain of TMV group.