It has recently been shown that tyrosyl radicals react with superoxide to form a peroxide adduct of tyrosine. Since myeloperoxidase oxidizes tyrosine to its radical, and neutrophils and monocytes contain myeloperoxidase as well as produce superoxide, we have investigated whether tyrosine peroxide could be a significant product of tyrosine oxidation by these cells. Oxidation of tyrosine by purified myeloperoxidase and a superoxide-generating system, and by stimulated human neutrophils, was found to generate peroxide adducts as detected in the xylenol orange (FOX) assay and by HPLC. Superoxide, hydrogen peroxide, and myeloperoxidase were required for formation of the peroxide. Dityrosine was also formed in each system, and in the presence of superoxide dismutase, suppression of tyrosine peroxide formation gave elevated formation of dityrosine. Quantitative estimates indicate that at physiological tyrosine concentration the peroxide is likely to be formed in preference to dityrosine and to be a significant product of neutrophils. This metastable peroxide therefore has the potential to contribute to neutrophil- or monocyte-mediated tissue injury.

Enzymatically generated tyrosyl radicals are effectively scavenged by reduced glutathione (GSH), thereby generating glutathione thiyl radicals and superoxide radicals, subsequently. Here, we have used horseradish peroxidase to generate tyrosyl radicals and investigated the fate of the superoxide radicals. At low GSH concentrations (with a maximum effect at 250 microM) a major reaction was between superoxide and the phenoxyl radical leading to a tyrosine peroxide. Formation of the peroxide was confirmed using a peroxide-specific colorimetric assay and detection of a new HPLC-peak. Its formation was inhibited by superoxide dismutase (SOD). The peroxide decomposed slowly in a reaction that was accelerated by GSH to give a new chromatographic peak. Increasing the GSH concentration decreased the amount of tyrosine peroxide formed and caused increases in rates of oxygen uptake and GSH oxidation. These increases were not seen in the presence of SOD and are consistent with GSH scavenging superoxide and leading to oxygen-dependent chain oxidation of GSH. Both pathways are undesirable for the cell and are effectively suppressed only if GSH as a radical scavenger acts in concert with SOD.

A full-length transcript, Imt1, encoding myo-inositol O-methyltransferase (EC 2.1.1.X) from the halophyte Mesembryanthemum crystallinum was expressed in Escherichia coli. The enzyme, IMT1, uses S-adenosyl-L-methionine to methylate myo-inositol to form D-ononitol. IMT1 with a monomeric mass of 41,000 was isolated by ammonium sulfate fractionation, gel filtration and ion exchange chromatography to apparent purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified recombinant enzyme was identical to that encoded by the cDNA sequence. The apparent Km for S-adenosylmethionine was 0.18 mM with a Vmax of 1550 pkat/mg protein. The Km for myo-inositol was 1.32 mM. The reaction became substrate-inhibited by concentrations of S-adenosylmethionine greater than 0.5 mM. Inositol methyltransferase was competitively inhibited 50% with 0.01 mM S-adenosyl-homocysteine, while 1 mM homocysteine, homoserine, or adenosine did not inhibit. The enzyme exhibited a pH optimum of 7.8 and a temperature optimum of 37°C. Activity of the isolated inositol methyltransferase was stable when stored at 4°C.

The influence of externally applied gaseous nitric oxide (NO) on spruce needles in relation to cGMP formation has been examined. Exposure of spruce needles (Picea abies) to gaseous nitric oxide leads to a strong and rapid increase of the cGMP concentration. Depending on the base level of cGMP in the untreated needles, the concentration of cGMP in the NO exposed needles increased up to four degrees of magnitude. The content of adenosine-3′,5′-cyclic monophosphate (cAMP) remained below the detection limit of the HPLC-method used (10− 7 moll− 1). Therefore, it was not subject to further investigation.

The effects of t-butyl hydroperoxide (tBOOH) on bovine liver catalase were investigated. tBOOH is accepted as a substrate of catalase and in the absence of hydrogen donors leads to a destruction of the enzyme via compound II formation. During the decomposition of this enzyme-substrate complex catalase serves as internal hydrogen donor which results in destruction of the enzyme. Evidence for this destruction is given by: a decrease of the Soret band in the uv/vis spectrum, iron release from the enzyme, decrease of the catalatic activity of the enzyme measured by oxygen release from hydrogen peroxide. Hydrogen donors like NADH and o-dianisidine have been found to protect the enzyme from destruction by tBOOH but lead to a structural alteration of the enzyme, shown by alteration of the electrophoretic mobility. In the presence of the hydrogen donor tBOOH is completely reduced to t-butanol, which is thought to proceed in a peroxidase-like reaction.

The described staining technique for catalase activity on polyacrylamide gel leads to sharp colourless bands on a uniform brown backgroud. It is based on the oxidation of a-dianisidine with hydrogen peroxide by the catalytic action of haemin. Catalase activity can be detected down to 0.25 U. The whole staining procedure needs about 30 min and parallel detection of peroxidase activity is possible.

Phenol polymerization via hydrogen peroxide and plant peroxidases is investigated. Hydrogen peroxide is generated via enzymatically reduced paraquat with ferredoxin reductase, NADPH and oxygen. In order to study the paraquat dependent generation of hydrogen peroxide an electrophoretic procedure is applied using wood peroxidase isoenzymes separated on polyacrylamide gels, and phenolic substrates as sensitive hydrogen peroxide detectors. To quantify this process gels are scanned with a laser densitometer. The theory of hydrogen peroxide formation by paraquat is further supported by photometrical detection of the complex II of horseradish peroxidase and the fluorometric measurement of oxidized homovanillic acid. The effects of superoxide dismutase and catalase are investigated; hydrogen peroxide formation is enhanced by the former and inhibited by the latter.