Hiroteru Sayo

Distribution of ubiquinone and ubiquinol homologues in rat tissues and subcellular fractions

The oxidized (UQox) and reduced (UQred) forms of ubiquinone (UQ) homologues in rat tissues and subcellular fractions were analyzed to elucidate their distribution and physiological role. UQ-9 and UQ-10 were detected in all tissues studied, and UQ-9 was the predominant homologue. The total amount of UQox-10 and UQred-10 was 20–50% that of UQox-9 and UQred-9. The levels of these homologues were highest in heart with lesser amounts occurring in kidney, liver and other organs. In liver and blood plasma, the UQred homologue amounted to 70–80% of the total UQ (UQox+UQred=t-UQ). UQred was less than 30% of t-UQ in other tissues and blood cells. t-UQ was much higher in leukocytes and platelets in blood than in erythrocytes. In erythrocytes, t-UQ was exclusively located in the cell membranes. UQox and UQred were also found in all subcellular fractions isolated from liver and kidney in about the same ratio as UQred/t-UQ was present in the whole organ. The levels of UQox and UQred per mg protein in subcellular fractions from liver were highest in mitochondria, with lesser amounts present in plasma membranes, lysosomes, Golgi complex, nuclei, microsomes and cytosol. In the mitochondria, the outer membranes were richer in t-UQ than the inner membranes. In the Golgi complex, the light and intermediate fractions were rich in t-UQ when compared to the heavy fraction. The possible physiological role of UQox and UQred in tissues and subcellular fractions is discussed.

Purification and some properties of peroxidases of rat bone marrow

Myeloperoxidase and eosinophil peroxidase were separated and purified from rat bone marrow cells using cetyltrimethylammonium bromide as the solubilizer and then with column chromatographies on CM-Sephadex C-50 and Con A-Sepharose. Both purified enzymes were observed to be apparently homogeneous by SDS-polyacrylamide gel electrophoresis. Myeloperoxidase consisted of two subunits of Mr 57,000 and 15,000, and eosinophil peroxidase two of 53,000 and 14,000. On structural analysis of the enzymes, their visual and ESR spectra revealed that the structure surrounding the heme in myeloperoxidase was different from that in eosinophil peroxidase. Moreover, substrate specificity and sensitivity to inhibitors such as azide and cyanide differed between the two enzymes. Rat bone marrow possesses two distinct peroxidases, myeloperoxidase and eosinophil peroxidase, which have different subunits and different heme microenvironments. Therefore, the difference in enzymatic function between the two peroxidases may be due to their structures.

The mechanism of myeloperoxidase-catalysed oxidation of aminopyrine

1. Myeloperoxidase catalysed the H2O2-supported oxidation of aminopyrine in the presence of Cl-, generating the aminopyrine cation radical (AP+.). The rate of AP+. formation was determined by monitoring the absorbance at 565 nm. The pH optimum of the reaction was around 5.0. The rate of AP+. formation increased with increasing concentration of aminopyrine. Inhibition by excess H2O2 was seen at pH 4.5-5.5. 2. When Cl- was replaced by Br-, the rate of AP+. formation increased significantly, and inhibition by H2O2 became less evident and was observed only at pH 5.5. The rate of chemical oxidation of aminopyrine by HOCl was much slower than that by Br2. 3. Compared with the chlorination of monochlorodimedone (MCD), the reaction between aminopyrine and HOCl, produced by the enzymic peroxidation of Cl-, is rate-limiting in the myeloperoxidase-catalysed oxidation of aminopyrine. The differences in kinetic behaviour between the myeloperoxidase-catalysed chlorination of MCD and oxidation of aminopyrine are explained by the low reactivity of HOCl towards aminopyrine.

Oxidative metabolism of propylthiouracil by peroxidases from rat bone marrow.

1. Propylthiouracil (PTU) was degraded by myeloperoxidase (MPO) or eosinophil peroxidase (EPO), purified from rat bone marrow, in the presence of H2O2 and Cl-. In the absence of either H2O2 or Cl-, MPO and EPO do not degrade PTU. Optimum concentrations of KCl for MPO and EPO were 50 and 250 mM, respectively. 2. The characteristics of PTU degradation by MPO-H2O2-Cl- were similar to those of the chlorinating activity of the peroxidase. 3. Hypochlorous acid as well as MPO-H2O2-Cl- also degraded PTU. Metabolites of PTU degradation by MPO-H2O2-Cl-, which were separated by C18 reversed phase h.p.l.c., were the same as those produced by hypochlorous acid. 4. Of the metabolites of PTU formed by MPO-H2O2-Cl-, one was identified as PTU sulphonic acid (6-propyl-4-hydroxypyrimidine-2-sulphonate) and another seemed to be propyluracil.

ESR studies on the oxidation of N,N-dimethyl-p-anisidine and its analogues catalyzed by myeloperoxidase

N,N-Dimethyl-p-anisidine (DMA) was used as a substrate to differentiate between the direct, or chloride-independent, and the indirect, or chloride-dependent, pathways characteristic of myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7). The chemical oxidation by sodium hypochlorite and the horseradish peroxidase-catalyzed oxidation by H2O2 were also investigated for a comparison. The chemical oxidation of DMA by NaOCl (DMA/NaOCl = 1) gave the p-N,N-dimethylaminophenoxy radical at pH 5 and 7. p-Benzoquinone and formaldehyde were determined as stable end-products. On the other hand, the cation radical of DMA was detected and p-benzoquinone was not obtained in the horseradish peroxidase-H2O2-Cl- system. In the presence of Cl- the myeloperoxidase-catalyzed oxidation at pH 5 gave nearly the same result as did the oxidation by NaOCl, whereas in the absence of Cl- the result of the oxidation was similar to that of the horseradish peroxidase-catalyzed oxidation, except for a low yield of formaldehyde formation, which was ascribed to the decomposition of H2O2 by the catalase activity of myeloperoxidase. Although the myeloperoxidase-catalyzed oxidation of DMA at pH 7 in the presence of Cl- gave only the cation radical of DMA, a fairly large amount of p-benzoquinone was obtained as a product. This result indicates that the indirect chloride-dependent oxidation is also operating at pH 7. The reaction mechanism for the myeloperoxidase-catalyzed oxidation of DMA is proposed.

Hydrogen peroxide-dependent oxidative metabolism of 1-methy1-2-mercaptoimidazole (methimazole) catalysed by myeloperoxidase

1. Myeloperoxidase catalysed the H2O2-supported oxidation of 1-methyl-2-mercaptoimidazole (MMI) in the presence of Cl-. The rate of MMI oxidation was determined by monitoring a decrease in the absorbance at 251 nm. The pH optimum of the oxidation was around 4.5. The rate of MMI oxidation showed typical Michaelis-Menten saturation kinetics with respect to H2O2. Inhibition by excess H2O2 was not seen. 2. When the H2O2/MMI ratio was 0.5, MMI was oxidized by hyochlorous acid produced by the myeloperoxidase-H2O2-Cl- system giving bis-(1-methyl-2-imidazolyl)disulphide (MMI-disulphide) as an initial product, which gradually underwent disproportionation and subsequent hydrolysis giving 1-methylimidazole and MMI as the final products stoichiometrically. 3. When the H2O2/MMI ratio was one or above, hypochlorous acid produced in excess reacted with MMI-disulphide to give unidentified compounds. The sum of the amounts of 1-methylimidazole formed and of MMI reformed was less than 81% of the MMI added.

Electrochemical immunoassay combined with column liquid chromatography: determination of phenytoin in human serum

A new electrochemical immunoassay combined with column liquid chromatography has been developed for the determination of phenytoin in human serum. Phenytoin was labelled with the electrochemically active nitroxide, and the separation of the free labelled antigen from other electrochemically active compounds in serum was accomplished by the use of gel chromatography. Serum samples were mixed with the antibody and the labelled antigen, incubated for 90 min, and then a 100-microliter aliquot of the mixture was directly injected to the column, which was equipped with an electrochemical detector. With 10 microliter of serum, the smallest detectable concentration of phenytoin was 2 micrograms/ml.