Yoshio Ushijima

Aliphatic l-α-hydroxyacid oxidase from rat livers purification and properties

Abstract l -α-Hydroxyacid oxidase has been purified 350-fold from a cytoplasmic extract of rat livers. Purification involved homogenization of rat livers, isolation of a light mitochondrial fraction, sonic treatment, ammonium sulfate fractionation, dialysis, chromatography of DEAE-cellulose, and gel-filtration through Sephadex G-zoo. The enzyme is specific for many l -aliphatic-α-hydroxy acids and glycolic acid and does not affect α-hydroxyisobutyric acid or aromatic hydroxy acids. Inhibition studies indicate that a sulfhydryl group is essential for enzyme activity. Diethyldithiocarbamate and KCN have been found to be powerful inhibitors of the enzyme which follow the kinetics of competititve and uncompetitive types, respectively. From the mode of actions of two inhibitors, typical metal binding and metal chelating agents on the enzyme, it seems likely that the enzyme does not require a metal cofactor. The spectrophotemetric studies of the highly purified enzyme and attempts to split off the prosthetic group have proved no evidence of functional flavin group or the NAD + . The reaction catalyzed by this enzyme is: The enzyme was found to be located principally in the light mitochondrial fraction of rat liver homogenates.

Identity of aliphatic l-α-hydroxyacid oxidase and glycolate oxidase from rat livers

Abstract l -α-Hydroxyacid oxidase and glycolate oxidase have been partially purified from rat livers and found to be identical, judging by substrate specificities, K m values for certain substrates and coenzyme (FMN), activation energy, inhibition rates by various reagents and pH optimum. K m values are as follows; glycolate, 2.4 × 10 −4 m ; l -α-hydroxyisocaproate, 1.26 × 10 −3 ; glyoxylate, 1.41 × 10 −4 m ; and FMN, 1.13 × 10 −6 m . K m values for glycolate and FMN are one-tenth and one-twentieth the literature values for hepatic glycolate oxidase. Sucrose density gradient centrifugation establishes that this enzyme is located in hepatic peroxisomes.

Chemiluminescence in L-tyrosine-H2O2-horseradish peroxidase system: Possible formation of tyrosine cation radical

Tyrosine-H2O2-horseradish peroxidase system at pH 7.4 emitted light in visible region. Phenolic compounds other than tyrosine were also emissive, whereas methoxy phenylalanine and phenyl compounds were not, in H2O2-peroxidase systems. Chemiluminescence spectrum of tyrosine of tyrosine-H2O2-horseradish peroxidase system showed two prominent peaks at 478 nm and 500 nm (Luminescence 1) and additional two or three peaks near 550 and 610 nm (Luminescence 2). Luminescence 1 is quite similar to the phosphorescence originated from an excited tyrosine in triplet state, while Luminescence 2 is quite similar to the phosphorescence originated from an indole in triplet state. Possible formation of tyrosine cation radical (a precursor of the excited tyrosine) and indole cation radical in the enzyme protein (a precursor of the excited tryptophan residue) were discussed.

Species difference in sensitivity to the diabetogenic action of triphenyltin hydroxide

The sensitivity to the diabetogenic action of triphenyltin hydroxide (TPTOH) was investigated in 5 species of experimental animals. A single oral administration of TPTOH produced marked hyperglycemia and triglyccridemia in rabbits and hamsters, but no evidence of diabetes was found in mice, rats and guinea-pigs. No morphological abnormality was observed in islet tissue from TPTOH-treated hamsters.

Degradation of thyroxine by the microsomal particles from rat liver: I. Correlation between thyroxine degradation and lipid peroxides

Abstract 1. 1. Particles which catalyze the degradation of l -thyroxine labeled with 131 I at the 3′ and 5′ positions in the β-ring have been isolated from rat liver microsomes and purified. 2. 2. The reaction requires Fe 2+ and protein present in microsomal particles. 3. 3. Thyroxine degradation in the above reaction is correlated with lipid peroxide formation. It may be, therefore, that the microsomal protein functions with unsaturated fatty acids present in the particles as a prosthetic group for the oxidation or hydroxylation of the β-phenyl ring of thyroxine.

Excitation of indole analogs by phagocytosing leukocytes

Abstract The system suspended with phagocytosing leukocytes and related system produce weak light which could be greatly amplified by indole analogs with plain fatty acids at 3 position. Main emitting species in indole-3-acetic acid or indole-3-propionic acid-sensitized system was analyzed spectrometrically in the dark and ascribed to the transition of an excited indole compound in triplet state to its ground state. Such an excited species would be generated by the oxidative way of the indole analogs but not through the dioxetane structure of 2 and 3 positions on indole ring.

No or little production of singlet molecular oxygen in HOCl or HOClH2O2 a model system for myeloperoxidase/H2O2/Cl−

Abstract The HOCl in chlorine-water oxidizes DPF to cis -DBE in parallel to the HOCl concentration. The addition of H 2 O 2 produces singlet molecular oxygen, and a bimol collision above pH 6.0, but not in the pH region 3.0 to 4.0. The DPF conversion to cis -DBE is initiated by a 1,2-position attack of OH − and Cl + , followed by the HCl elimination. The oxidation potency of HOCl is much greater than the singlet molecular oxygen generated in chlorine-water/H 2 O 2 solution, on the pH range 6.0 to 8.0 where both HOCl and OCl − are present.

Triphenyltin chloride inhibits superoxide production by human neutrophils stimulated with a surface active agent.

Treatment of human neutrophils with triphenyltin chloride (TPTCl)‐inhibited superoxide (O− 2) production stimulated with phorbol myristate acetate (PMA). TPTCl was more potent as inhibitor of O− 2 production than other phenyltin compounds. The O− 2 production by the xanthine oxidase—acetaldehyde system was not inhibited by TPTCl. This finding indicates that TPTCl does not itself react with O− 2. Furthermore, TPTCl did not influence the isolated NADPH oxidase at all, though O− 2 production of neutrophils stimulated with PMA in the presence of TPTCl was inhibited. These results indicate that TPTCl inhibits the activation process of the O− 2 generating system.

Aliphatic l-α-hydroxyacid oxidase from rat liver: II. A flavoprotein

Abstract An aliphatic l -α-hydroxyacid oxidase present in a light mitochondrial fraction of rat liver cells was purified some 600-fold. At this stage the enzyme had a purity of 67%. The enzyme was still contaminated with catalase, and its absorption spectrum did not show a typical flavoprotein curve. Absorbance at 460 mμ was rapidly reduced by the addition of dithionite in air or of substrate during anaerobiosis. The difference spectrum of the reduced enzyme suggested that the enzyme is a flavoprotein. Spectrophotometry and paper chromatography of the extract of the enzyme after heat treatment indicated that the prosthetic group is FMN.

Chemiluminescence from human polymorphonuclear leukocytes activated with opsonized zymosan.

To prove the mechanism of photon emission during activation of leukocytes, a model system of human polymorphonuclear leukocytes (PMNs)-opsonized zymosan (OZ)-tyrosine (or none) or myeloperoxidase (MPO)-H2O2-tyrosine was employed, and three parameters-chemiluminescence yield and intensity, a metabolite such as bityrosine (BT), and chemiluminescence spectra-were studied. With the PMN system, the luminescence was enhanced by addition of tyrosine, its analogues, or albumin, but was inhibited by hydroxyurea, superoxide dismutase (SOD), or NaN3 (an inhibitor of MPO), indicating participation of tyrosine phenoxyl radicals, O2.- and MPO in the luminescence. With the PMN-OZ-tyrosine system, chemiluminescence yield was parallel to the BT formation. These results were essentially the same as those obtained with the MPO-H2O2-tyrosine system, except that luminescence from the latter system was not inhibited by SOD. When human albumin was exposed to the MPO-H2O2 system, BT was detected after hydrolysis of the protein in the mixture. Judging from the chemiluminescence spectra of activated PMNs and the MPO-catalyzed tyrosine oxidation, at least two excited species in triplet states-one for tyrosine and another for BT-would be generated in these systems. The luminescence may originate from the reaction of tyrosine phenoxyl radicals (cation radicals) with O2.- and/or peroxidase compound III (Fe...IIIO2.-).