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A J Kettle

Publications and source records attributed to A J Kettle.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.

Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 microM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which subsequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.

Aniline Compounds↗
PubMed1996

Neutrophils convert tyrosyl residues in albumin to chlorotyrosine.

Hypochlorous acid chlorinates tyrosyl residues in small peptides to produce chlorotyrosine. Detection of chlorotyrosine has the potential to unequivocally identify the contribution hypochlorous acid makes to inflammation. I have developed a selective and sensitive HPLC assay for measuring chlorotyrosine. When albumin was exposed to reagent hypochlorous acid, or that produced by myeloperoxidase and stimulated neutrophils, tyrosyl residues in the protein were converted to chlorotyrosine. About 2% of the hypochlorous acid generated by neutrophils was accounted for by the formation of chlorotyrosine. These results demonstrate that chlorotyrosine will be a useful marker for establishing a role for hypochlorous acid in host defence and inflammation.

Amino Acid Sequence↗
PubMed1996

Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.

Azides↗
PubMed1995

Chlorination of tyrosyl residues in peptides by myeloperoxidase and human neutrophils.

Hypochlorous acid is the major strong oxidant generated by human neutrophils, and it has the potential to cause much of the tissue damage that these inflammatory cells promote. It is produced from hydrogen peroxide and chloride by the heme enzyme myeloperoxidase. To unequivocally establish that hypochlorous acid contributes to inflammation, a stable and unique marker for its reaction with biomolecules needs to be identified. In this investigation we have found that reagent hypochlorous acid reacts with tyrosyl residues in small peptides and converts them to chlorotyrosine. Purified myeloperoxidase in combination with hydrogen peroxide and chloride, as well as stimulated human neutrophils, chlorinated tyrosine in the peptide Gly-Gly-Tyr-Arg. Rather than reacting directly with the aromatic ring of tyrosine, hypochlorous acid initially reacted with an amine group of the peptide to form a chloramine. The chloramine then underwent an intramolecular reaction with the tyrosyl residue to convert it to chlorotyrosine. This indicates that tyrosyl residues in proteins that are close to amine groups will be susceptible to chlorination. Peroxidases are the only enzymes capable of chlorinating an aromatic ring. Furthermore, myeloperoxidase is the only human enzyme that produces hypochlorous acid under physiological conditions. Therefore, chlorotyrosine will be a specific marker for the production of hypochlorous acid in vivo and for the involvement of myeloperoxidase in inflammatory tissue damage.

Amino Acid Sequence↗
PubMed1995

Inhibition of myeloperoxidase by benzoic acid hydrazides.

Myeloperoxidase is the most abundant protein in neutrophils and catalyses the conversion of H2O2 and chloride into HOCl. To help clarify the role of this enzyme in bacterial killing and inflammation, a specific and potent inhibitor needs to be identified. We have studied a series of benzoic acid hydrazides and found that in general they inhibit the peroxidation activity of myeloperoxidase with an IC50 value of less than 10 microM. The IC50 values of derivatives with substituents containing oxygen or nitrogen were related to their Hammett substituent constants. This indicates that myeloperoxidase oxidizes the hydrazide group of these compounds, and the degree to which they inhibit the enzyme is dependent on the ease of their oxidation. Unsubstituted benzoic acid hydrazide and its 4-chloro derivative were poor inhibitors of peroxidation. Thus it is likely that hydrogen-bonding of the enzyme to substituents containing oxygen or nitrogen increases the binding affinity of the hydrazides and enhances their oxidation by myeloperoxidase. 4-Aminobenzoic acid hydrazide (ABAH) was the most potent inhibitor of peroxidation. It irreversibly inhibited HOCl production by the purified enzyme, having an IC50 value of 0.3 microM. With neutrophils stimulated with opsonized zymosan or phorbol myristate acetate, ABAH inhibited HOCl production by up to 90% and the IC50 values were 16 microM and 2.2 microM respectively. In the presence of superoxide dismutase, these values decreased to 6.4 microM and 0.6 microM respectively. ABAH had no effect on superoxide radical (O2-.) production and degranulation by neutrophils, nor did it inhibit catalase or glutathione peroxidase. Thus ABAH is an effective and selective inhibitor that should be useful for determining the contribution of myeloperoxidase to oxidant-mediated reactions of neutrophils.

Aniline Compounds↗
PubMed1995

Myeloperoxidase oxidizes mitoxantrone to metabolites which bind covalently to DNA and RNA.

The anticancer agent mitoxantrone is readily oxidized by the human haem enzyme myeloperoxidase and hydrogen peroxide. Intercalation of mitoxantrone with DNA inhibited oxidation of the drug by myeloperoxidase. However, at a physiological ionic strength, significant oxidation of the drug was evident. At a H2O2:mitoxantrone ratio of 1.0, myeloperoxidase oxidized mitoxantrone to a metabolite (product B) which associated reversibly with DNA. At greater hydrogen peroxide concentrations, two further metabolites were produced (products C and D), neither of which associated reversibly with DNA, as indicated by the absence of any spectral change in the presence of DNA. Long exposure of the products derived from the oxidation of [14C]mitoxantrone by myeloperoxidase resulted in a time-dependent covalent binding of the activated drug to both DNA and RNA. The amount of DNA adduct increased linearly with the extent of oxidation of mitoxantrone (up to a H2O2:mitoxantrone ratio of 5.0). No adducts resulted from exposure of the oxidized product B to DNA, but adducts formed following further oxidation of B by myeloperoxidase. The myeloperoxidase-catalysed oxidation of mitoxantrone to products capable of interacting covalently and non-covalently with nucleic acids may represent an important mode of action of mitoxantrone against acute myeloid leukemias since these cells (including neutrophils, monocytes and their precursors) contain high levels of myeloperoxidase.

Antineoplastic Agents↗
PubMed1994

Oxidative metabolism of mitoxantrone by the human neutrophil enzyme myeloperoxidase.

The anti-cancer drug mitoxantrone is readily oxidized by the human heme enzyme myeloperoxidase (MPO) and H2O2. Direct oxidation yielded up to three products, which depended on the ratio of H2O2 to mitoxantrone. At an H2O2: mitoxantrone ratio of 1.0, one major product was obtained, with a spectrum and HPLC retention time identical to that resulting from oxidation by horseradish peroxidase. This metabolite is a substituted hexahydronaphtho[2,3-f]quinoxaline-7,12-dione and has been discovered in the urine of patients treated with mitoxantrone, hence implicating MPO in the in vivo metabolism of mitoxantrone. At higher concentrations of H2O2, the oxidation of mitoxantrone was more complex, with two further metabolites being identified. When mitoxantrone was incubated with neutrophils that had been stimulated with phorbol myristate acetate, it was oxidized by an MPO-dependent mechanism. Therefore, it appears that MPO may play a significant role in the clinical activity displayed by mitoxantrone against acute myelogenous leukemias, as neutrophils, monocytes and their bone marrow precursors contain high levels of the enzyme.

Chromatography, High Pressure Liquid↗
PubMed1994

Superoxide-dependent hydroxylation by myeloperoxidase.

When stimulated, neutrophils undergo a respiratory burst converting oxygen to superoxide. Although superoxide is critical for microbial killing by phagocytic cells, the precise role it plays has yet to be established. It has been proposed to optimize their production of hypochlorous acid and to be required for the generation of hydroxyl radicals. Superoxide is also involved in the hydroxylation of salicylate by neutrophils. However, the mechanism of this reaction is unknown. We found that neutrophils stimulated with opsonized zymosan hydroxylated salicylate to produce mainly 2,5-dihydroxybenzoate. Its formation was dependent on superoxide and a heme protein but was independent of hydrogen peroxide and hydroxyl radicals. Production of 2,5-dihydroxybenzoate was enhanced by methionine, which scavenges hypochlorous acid. Neutrophils from an individual with myeloperoxidase deficiency hydroxylated salicylate at only 13% of the level of control cells. Purified human myeloperoxidase and xanthine oxidase plus hypoxanthine hydroxylated salicylate to produce 2,5-dihydroxybenzoate. As with neutrophils, the reaction required superoxide but not hydrogen peroxide and was unaffected by hydroxyl radical scavengers. Thus, myeloperoxidase catalyzes superoxide-dependent hydroxylation. This newly recognized reaction may be relevant to the in vivo functions of superoxide and myeloperoxidase.

Humans↗
PubMed1994

Assays using horseradish peroxidase and phenolic substrates require superoxide dismutase for accurate determination of hydrogen peroxide production by neutrophils.

We used horseradish peroxidase and either scopoletin, homovanillic acid, or phenol red to measure hydrogen peroxide generated by human neutrophils. With these assays, superoxide dismutase significantly increased the amount of hydrogen peroxide detected. In contrast, it had no effect when the accumulation of hydrogen peroxide was measured with a hydrogen peroxide electrode. We propose that superoxide interferes with horseradish peroxidase-dependent assays so that hydrogen peroxide is underestimated. Thus, when using these assays, superoxide dismutase must be added to neutrophils to ensure that all the hydrogen peroxide they produce is detected.

Horseradish Peroxidase↗
PubMed1993

Superoxide is an antagonist of antiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase.

Myeloperoxidase, the most abundant enzyme in neutrophils, catalyses the conversion of hydrogen peroxide and chloride to hypochlorous acid. This potent oxidant has the potential to cause considerable tissue damage in many inflammatory diseases. We have investigated the ability of dapsone, diclofenac, primaquine, sulfapyridine and benzocaine to inhibit hypochlorous acid production by stimulated human neutrophils. The drugs were also tested against purified myeloperoxidase using xanthine oxidase to generate hydrogen peroxide and superoxide. The inhibitory effects of the drugs on hypochlorous acid production, either by cells stimulated with phorbol myristate acetate or by myeloperoxidase and xanthine oxidase, were significantly less than those determined with myeloperoxidase and reagent hydrogen peroxide. Comparable potency was observed only when superoxide dismutase was present to remove superoxide. We also observed that with the xanthine oxidase system, inhibition of hypochlorous acid production by dapsone decreased markedly as the concentration of myeloperoxidase increased. Dapsone was a poor inhibitor of hypochlorous acid production by neutrophils stimulated with opsonized zymosan, regardless of the presence of superoxide dismutase. With this phagocytic stimulus, catalase inhibited hypochlorous acid formation by only 60%, which indicates that a substantial amount of the hypochlorous acid detected originated from within phagosomes. Thus, it is apparent that dapsone is unable to affect intraphagosomal conversion of hydrogen peroxide to hypochlorous acid. All the drugs inhibit myeloperoxidase reversibly by trapping it as its inactive redox intermediate, compound II. We propose that superoxide limits the potency of the drugs by reducing compound II back to the active enzyme. Furthermore, under conditions where the activity of myeloperoxidase exceeds that of the hydrogen peroxide-generating system, which is most likely to occur in phagosomes, partial inhibition of myeloperoxidase need not affect hypochlorous acid production. We conclude that drugs that inhibit myeloperoxidase by converting it to compound II are unlikely to be effective against hypochlorous acid-mediating tissue damage.

Anti-Inflammatory Agents↗
PubMed1992

Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase.

Oxidative metabolism of the anti-cancer drug amsacrine 4'-(9-acridinylamino) methane-sulphan-m-anisidide has been suggested to account for its cytotoxicity. However, enzymes capable of oxidizing it in non-hepatic tissue have yet to be identified. A potential candidate, that may be relevant to the metabolism of amsacrine in blood and its action in myeloid leukaemias and myelosuppression, is the haem enzyme myeloperoxidase. We have found that the purified human enzyme oxidizes amsacrine to its quinone diimine, either directly or through the production of hypochlorous acid. In comparison, the 4-methyl-5-methylcarboxamide derivative of amsacrine, CI-921 9-[[2-methoxy-4[(methylsulphonyl)-amino]phenyl]amino)-N, 5-dimethyl-4-acridine carboxamide, reacted poorly with myeloperoxidase, although it was oxidized by hypochlorous acid. Detailed studies of the mechanism by which myeloperoxidase oxidizes amsacrine revealed that the semiquinone imine free radical is a likely intermediate in this reaction. Oxidation of amsacrine analogues indicated that factors other than their reduction potential determine how readily they are metabolized by myeloperoxidase. Both amsacrine and CI-921 inhibited production of hypochlorous acid by myeloperoxidase. CI-921 acted by trapping the enzyme as the inactive redox intermediate compound II. Amsacrine inhibited by a different mechanism that may involve conversion of myeloperoxidase to compound III, which is also unable to oxidize Cl-. The susceptibility of amsacrine to oxidation by myeloperoxidase indicates that this reaction may contribute to the cytotoxicity of amsacrine toward neutrophils, monocytes and their precursors.

Amsacrine↗
PubMed1992

Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone.

Myeloperoxidase (MPO) is a prime candidate for mediating the inflammatory tissue damage of neutrophils because it converts Cl- to the potent oxidant hypochlorous acid. It also oxidizes xenobiotics to reactive free radicals. We have found that the kinetics of oxidation of hydroquinone by myeloperoxidase are inadequately explained by the classical peroxidase mechanism. Peroxidation of hydroquinone displayed a distinct lag phase, which was practically abolished by excluding O2 and was eliminated by adding benzoquinone at the start of the reaction. Superoxide dismutase increased the rate of peroxidation by 40% but did not eliminate the lag phase. Spectral investigations revealed that during the initial phase of the reaction, MPO was converted to oxy-MPO, or compound III, by a mechanism that was not reliant on superoxide. Benzosemiquinone, however, was able to convert ferric-MPO to compound III. Both compound III and ferro-MPO reacted with benzoquinone to regenerate ferric-MPO. We propose that the lag phase occurs because benzosemiquinone reduces ferric-MPO to ferro-MPO, which rapidly binds O2 to form compound III. Since compound III is outside the peroxidation cycle, conversion of hydroquinone to benzoquinone is retarded. However, as benzoquinone accumulates, it oxidizes ferro-MPO and compound III to ferric-MPO, thereby increasing the rate of peroxidation. There is a minimal lag phase under an atmosphere of N2 because ferro-MPO would be rapidly oxidized by benzoquinone, without formation of compound III. We conclude that when substrates produce radicals capable of reducing ferric-MPO, they will be peroxidized efficiently only if oxy-MPO is readily recycled. Furthermore, these radicals will prevent MP3+ from reacting with H2O2, and thereby prevent the enzyme from oxidizing Cl- to hypochlorous acid. Thus, this mechanism could be exploited to prevent hypochlorous acid-mediated inflammatory tissue damage.

Animals↗
PubMed1991

Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs.

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils, and should therefore be expected to contribute to the damage caused by these inflammatory cells. It is produced from H2O2 and Cl- by the heme enzyme myeloperoxidase (MPO). We used a H2O2-electrode to assess the ability of a variety of anti-inflammatory drugs to inhibit conversion of H2O2 to HOCl. Dapsone, mefenamic acid, sulfapyridine, quinacrine, primaquine and aminopyrine were potent inhibitors, giving 50% inhibition of the initial rate of H2O2 loss at concentrations of about 1 microM or less. Phenylbutazone, piroxicam, salicylate, olsalazine and sulfasalazine were also effective inhibitors. Spectral investigations showed that the inhibitors acted by promoting the formation of compound II, which is an inactive redox intermediate of MPO. Ascorbate reversed inhibition by reducing compound II back to the active enzyme. The characteristic properties that allowed the drugs to inhibit MPO reversibly were ascertained by determining the inhibitory capacity of related phenols and anilines. Inhibition increased as substituents on the aromatic ring became more electron withdrawing, until an optimum reduction potential was reached. Beyond this optimum, their inhibitory capacity declined. The best inhibitor was 4-bromoaniline which had an I50 of 45 nM. An optimum reduction potential enables inhibitors to reduce MPO to compound II, but prevents them from reducing compound II back to the active enzyme. Exploitation of this optimum reduction potential will help in targeting drugs against HOCl-dependent tissue damage.

Aminobenzoates↗
PubMed1991

The influence of superoxide on the production of hypochlorous acid by human neutrophils.

Human neutrophils stimulated with opsonized zymosan promoted hypochlorous acid (HOCl)-dependent loss of monochlorodimedon. Formation of HOCl was completely inhibited by catalase, and it was also inhibited up to 70% by SOD. There was no inhibition by desferal, DTPA, mannitol or dimethylsulphoxide, which excluded the involvement of .OH. Our results indicate that generation of O2- by neutrophils enables these cells to enhance their production of HOCl. Furthermore, inhibition of neutrophil processes by SOD and catalase does not necessarily implicate .OH. We propose that O2- may potentiate oxidant damage at inflammatory sites by boosting the myeloperoxidase-dependent production of HOCl.

Catalase↗
PubMed1990

Superoxide enhances hypochlorous acid production by stimulated human neutrophils.

Stimulated neutrophils undergo a respiratory burst discharging large quantities of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and Cl- to hypochlorous acid. Human neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in Cl(-)-free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalase, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorbol myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.

Catalase↗
PubMed1989

Influence of superoxide on myeloperoxidase kinetics measured with a hydrogen peroxide electrode.

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.

Chlorides↗