Myeloperoxidase-oxidase oxidation of cysteamine.

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.     

Role of thiols in degradation of proteins by cathepsins.

The effects of thiols on the breakdown of 125I-labelled insulin, albumin and formaldehyde-treated albumin by highly purified rat liver cathepsins B, D, H and L at pH 4.0 and 5.5 were studied. At both pH values degradation was strongly activated by the thiols cysteamine, cysteine, dithiothreitol, glutathione and 2-mercaptoethanol, and its rate increased with increasing thiol concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect concentration. Preincubation of the protein substrates with 5 mM-glutathione did not affect the rate of degradation by cathepsin D or L, and determination of free thiol groups after incubation of the proteins in the presence of glutathione but without cathepsin showed that their disulphide bonds were stable under the incubation conditions. Sephadex G-75 chromatography of the acid-soluble products of insulin digestion by cathepsin D or L suggested that thiols can reduce disulphide bonds in proteins after limited proteolysis. The resultant opening-up of the protein structure would lead to further proteolysis, so that the two processes (proteolysis and reduction) may act synergistically. By using the osmotic protection method it was shown that, at a physiological pH, cysteamine, and its oxidized form cystamine, can cross the lysosome membrane and thus may well be the physiological hydrogen donor for the reduction of disulphides in lysosomes. The results are discussed in relation to the lysosomal storage disease cystinosis.     

Cysteamine Oxygenase: Possible Involvement of Superoxide Ion in the Catalytic Mechanism

The reaction catalyzed by cysteamine oxygenase on cysteamine in the presence of phenazine methosulphate as cofactor like compound is inhibited by nitroblue tetrazolium, a scavenger of superoxide ions. The reaction is not inhibited by superoxide dismutase and allyl alcohol and it is not activated by superoxide ions produced in solution. Nitroblue tetrazolium is reduced by cysteamine or mercaptoethanol and phenazine methosulphate. This reaction is completely inhibited by superoxide dismutase. In the presence of cysteamine oxygenase the reduction with mercaptoethanol is greatly enhanced and it is only partially inhibited by superoxide dismutase. According to these data a reaction mechanism is proposed in which superoxide ions and thiyl radicals are produced at the active site during catalysis.     

Involvement of cysteine, serotonin and their analogues in peroxidase-oxidase reactions

Myeloperoxidase-oxidase reactions with close to physiological concentrations of thiols and phenols were studied. Cysteine was shown to be a myeloperoxidase-oxidase substrate when catalytic amounts of serotonin were added as cosubstrate. Penicillamine could be substituted for cysteine and acetaminophen could be substituted for serotonin. The properties of these peroxidase-oxidase reactions, e.g. the dependence on substrate and myeloperoxidase concentration, reduced oxygen species, metal ions and pH, were studied. Also, eosinophil, lacto- and horseradish peroxidase could catalyse these reactions.     

Factors influencing the oxidation of cysteamine and other thiols: implications for hyperthermic sensitization and radiation protection

Some of the factors influencing the oxygen uptake and peroxide formation for cysteamine (MEA) and other thiols in serum-supplemented modified McCoy's 5A, a well-known medium used to cultivate a variety of cells in vitro, have been studied. The oxidation of MEA and cysteine in modified McCoy's 5A has been compared with that in Ham's F-12, MEM, and phosphate-buffered saline. All of the growth media were supplemented with 10% calf serum and 5% fetal calf serum. The rate of oxygen uptake for all of the studied thiols was greatest in McCoy's 5A. The data indicate that this medium may contain more copper than the other preparations. MEA and cysteine were found to be more effective at 0.4 mM at producing peroxide than dithiothreitol (DTT). N-acetylcysteine was the least reactive. The ability to produce peroxide is dependent upon the temperature, the concentration of thiol, the presence of copper ions, and pH of the medium. MEA and other thiol oxidation is inhibited by the copper chelator diethyldithiocarbamate. Catalase also reduces the oxygen uptake for all thiols. This inhibition involves the recycling of peroxide to oxygen. Superoxide dismutase (SOD) was found to stimulate the oxygen uptake in the case of MEA and cysteine, but had little or no effect with DTT and glutathione. The combined presence of SOD and catalase resulted in less inhibition of oxygen uptake than that obtained by catalase alone. Alkaline pH was found to enhance the oxidation of cysteine and MEA. An important observation was the inhibition of MEA oxidation at 0 degrees C and the stimulation at 42 degrees C. The results indicate that many problems may arise when thiols are added to various media. A major consideration is concerned with the production of peroxide, superoxide, and reduced trace metal intermediates. The presence of these intermediates may result in the production of hydroxyl radical intermediates as well as the eventual oxygen depletion from the medium. Oxygen depletion may alter the results of radiation sterilization and carcinogen activation. Radical production will cause cell damage that is temperature dependent. Therefore, careful consideration must be given to changes in oxygen tension when thiols are added to cells growing in complicated growth medium to protect against either chemical or radiation damage.     

Synergism between substrate and non-substrate thiols in peroxidase-oxidase reactions

Cysteamine and reduced glutathione were shown to act synergistically as peroxidase-oxidase substrates as measured by oxygen consumption and Nitro Blue Tetrazolium reduction. Cysteine methyl ester could be substituted for cysteamine and N-acetylcysteine and penicillamine could be substituted for glutathione. The involvement of reduced oxygen species and the effects of pH and chloride were studied. A possible mechanism of peroxidase-oxidase oxidation of cysteamine and glutathione is proposed. These studies show that peroxidase oxidase reactions can occur with close to physiological concentrations of peroxidase and thiols.     

Study of activated oxygen production by some thiols using chemiluminescence.

2-3-dimercapto-1-propane sulfonic acid, D-penicillamine and meso-dimercapto succinic acid, drugs widely applied as antidota against metal poisoning, and cysteine and glutathione were studied with respect to their ability to generate and to scavenge superoxide anion radical. Superoxide production and scavenging were tested by means of luminol-dependent chemiluminescence. In presence of 1 mumol/l ADP-Fe3+ only cysteine and meso-dimercapto succinic acid induced chemiluminescence which could be inhibited by superoxide dismutase. 2,3-dimercapto-1-propane sulfonic acid, D-penicillamine and glutathione acted as O2- scavengers. These thiols inhibited O2(-)-dependent lipid peroxidation thus acting as antioxidants, whereas cysteine and meso-dimercapto succinic acid accelerated peroxidation. It is suggested that the toxic side effects of thiols may be due to their ability to generate or to scavenge free radicals.     

Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide.

The reactivities of glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril with superoxide generated from xanthine oxidase and hypoxanthine, and with reagent hydrogen peroxide, have been investigated. Rates of thiol loss on adding hydrogen peroxide, and superoxide-dependent thiol loss and oxygen uptake were measured. The relative reactivities of the different thiols with both oxidants were inversely related to the pK of the thiol group, such that at pH 7.4, penicillamine was the most reactive. N-acetylcysteine weakly reactive and no reaction was seen with captopril. For hydrogen peroxide, the calculated rate constants for the reaction with the thiolate anion all fell within the range 18-26 M(-1) s(-1). With superoxide, our results are consistent with each thiol reacting via a short chain that consumes oxygen and regenerates superoxide. Only with some of the thiols, was the consumed oxygen recovered as hydrogen peroxide. Reported values for the rate constant for the reaction of thiols with superoxide vary over four orders of magnitude, with the highest being > 10(5) M(-1) s(-1). Due to the complexity of the chain reaction, no study so far has been able to obtain accurate values and we consider the best estimates to be in the 30 to 1000 M(-1) s(-1) range.     

Interactions between metals, ligands, and oxygen in the autoxidation of 6-hydroxydopamine: mechanisms by which metal chelation enhances inhibition by superoxide dismutase

Transition metal ions and superoxide participate in different autoxidations to a variable extent. In the reaction of 6-hydroxydopamine (6-OHDA) with oxygen at pH 7.0 or 8.0, addition of 5 to 300 U/ml superoxide dismutase inhibited autoxidation by up to 96% at the highest concentrations. Superoxide dismutase at concentrations of 5-20 U/ml inhibited by less than 40% when present alone, but inhibited by over 99% in the presence of desferrioxamine or histidine. EDTA also enhanced the inhibition by 20 U/ml superoxide dismutase to 86%, even though EDTA accelerated the autoxidation of 6-OHDA when present alone or with desferrioxamine. In contrast, other ligands, such as ADP or phytic acid, had little or no effect on inhibition by superoxide dismutase. Proteins such as albumin, cytochrome oxidase, or denatured superoxide dismutase also enhanced inhibition by active superoxide dismutase from less than 40% to over 90%. Evidently, in the presence of redox active metals, autoxidation occurs by inner sphere electron transfer, presumably within a ternary 6-OHDA.metal.oxygen complex. This mechanism does not involve free O2-. and is not inhibited by superoxide dismutase. On the other hand, the presence of certain ligands (including proteins) diminishes the ability of trace metals to exchange electrons with 6-OHDA or oxygen by an inner sphere mechanism. These ligands render autoxidation dependent on propagation by O2-. and therefore inhibitable by superoxide dismutase. Previously conflicting reports that superoxide dismutase alone inhibits 6-OHDA autoxidation are thus explicable on the basis that at sufficient concentration the apoprotein coordinates trace metals in such a way to preclude inner sphere metal catalysis.