Shozo Yamamoto

Selective inhibition of prostaglandin endoperoxide thromboxane isomerase by 1-carboxyalkylimidazoles

1-Carboxyalkylimidazoles inhibited the conversion of prostaglandin H2 to thromboxane B2 and 12L-hydroxy-5,8,10-heptadecatrienoic acid by a partially purified enzyme (prostaglandin endoperoxide thromboxane isomerase) from bovine platelet microsomes. The degree of the inhibition was dependent on the length of carboxyalkyl chain. 1-Carboxyheptylimidazole was the most potent inhibitor, and an almost complete inhibition was obtained at a concentration on the order of 1 micron. The inhibition, as examined with 1-carboxyheptylimidazole, was of noncompetitive type. These 1-carboxyalkylimidazoles did not affect the formation of prostaglandin H2 from arachidonic acid. Such a selective inhibition was also demonstrated by the reaction of bovine platelet microsomes with arachidonic acid in the presence of 1-carboxyheptylimidazole, resulting in the accumulation of prostaglandin H2 as an intermediate. Furthermore, a series of 1-alkylimidazoles with no carboxyl group also inhibited the isomerase at higher concentrations. However, the inhibition was not specific for the isomerase; namely, the prostaglandin H2 formation from arachidonic acid was also affected.

Isolation of an activator for prostaglandin hydroperoxidase from bovine vesicular gland cytosol and its identification as uric acid

Abstract The enzymatic conversion of prostaglandin G 1 to H 1 was stimulated by an activator present in the cytosol of bovine vesicular gland. The activator was purified by Sephadex G-25 gel filtration and Dowex 1 column chromatography. The purified activator was identified to be uric acid by thin layer chromatography, ultraviolet and infrared absorption spectroscopy and combined gas chromatography-mass spectroscopy. Among various purine compounds tested, only uric acid and 2,8-dihydroxyadenine were active.

Reactions of prostaglandin endoperoxides with prostaglandin I synthetase solubilized from rabbit aorta microsomes

Abstract Prostaglandin I (prostacyclin) synthetase was solubilized from rabbit aorta microsomes. When the solubilized enzyme was reacted with prostaglandin H2 and the reaction mixture was extracted under acidic conditions, the major product was 6-keto-prostaglandin F1α. The formation of prostaglandin I2 as the primary reaction product was demonstrated by the treatment of the reaction mixture with diazomethane under non-acidic conditions. In contrast, the reaction of prostaglandin H1 with the enzyme resulted in the production of 12L-hydroxy-8,10-heptadecadienoic acid rather than prostaglandin I1.

Inhibitory effects of superoxide dismutases and various other proteins on the nitroblue tetrazolium reduction by phagocytizing guinea pig polymorphonuclear leukocytes.

Summary The reduction of nitroblue tetrazolium by guinea pig peritoneal polymorphonuclear leukocytes was studied during the phagocytosis of latex particles. Superoxide dismutases from various sources inhibited the reduction of the dye. However, almost the same degree of inhibition was observed with a number of other proteins including albumins, γ-globulin, histone and trypsin inhibitor, as well as with the heat inactivated dismutases. The results suggest a non-specific inhibitory effect of superoxide dismutase proteins on the reduction of nitroblue tetrazolium rather than a specific enzymic inhibition of the superoxide-mediated reduction of the dye.

Human platelet aggregation induced by prostaglandin endodisulfide

The effect on human platelet functions of 9,11-dithio analogues of prostaglandin endoperoxide was investigated. Methyl (5Z, 9alpha, 11alpha, 13E, 15S)-9,11-epidithio-15-hydroxyprosta-5,13-dienoate induced platelet aggregation, while the 9beta,11beta-epimer was inactive. The platelet aggregation caused by the 9alpha,11alpha-dithio analogue was associated with serotonin release from platelets, and was inhibited by methyl ester of prostaglandin I2 (prostacyclin) but not by indomethacin.

Studies on the reaction specificity of the flavoprotein lysine monooxygenase with modified substrates

Abstract Various modified substrates of lysine monooxygenase were examined to determine whether they were oxygenated or oxidized. Among various methyllysines tested, N ϵ - and δ-methyllysine underwent predominantly an oxygenative decarboxylation, producing the corresponding acid amides, while γ-methyllysine underwent predominantly an oxidative deamination with an α-keto acid as the reaction product. β-Methyllysine was inactive as substrate. All four methyllysines decreased the cooperativity of the enzyme with the normal substrate, lysine. Furthermore, lysine oxygenation was competitively inhibited by all of them except for β-methyllysine, which was much less inhibitory than the other methyllysines. Other analogs with a chloro or hydroxyl group at either the δ or the γ position were both oxygenated and oxidized. Analogs with a modified carboxyl or α-amino group were inactive as substrates.

ENZYMOLOGICAL STUDIES ON PROSTAGLANDIN BIOSYNTHESIS

Prostaglandin (PG) synthetase system was localized in bovine vesicular gland microsomes, which produced PGE 1 from 8,11,14-eicosatrienoic acid. Three compounds (heme, tryptophan and glutathione) were required as activators. The enzyme system was solubilized from the microsomes and separated into two enzyme components (Fractions I and II). Simultaneous presence of both fractions caused the production of PGE 1 .

A dehydrogenase reaction catalyzed by lysine monooxygenase, a flavooxygenase.

Abstract Lysine monooxygenase is a pseudomonad flavoprotein which, in the presence of molecular oxygen, catalyzes either oxygenative decarboxylation or oxidative deamination of amino acid substrates. Lysine and arginine are predominantly oxygenated, while ornithine and alanine only in combination with propylamine are oxidized. In the absence of oxygen, however, the enzyme-catalyzed dehydrogenation of lysine, arginine, ornithine and alanine, when ferricyanide and phenazine methosulfate were supplied as a terminal electron acceptor and an intermediate electron carrier, respectively. The dehydrogenated product from each amino acid was identified to be the corresponding α-keto acid by paper electrophoresis, thin-layer chromatography and chemical degradation. The stoichiometry of the amino acid consumption, the α-keto acid formation and the ferricyanide reduction was approximately 1:1:2 with each amino acid. The dehydrogenase activity toward oxidase-type substrates was higher than that toward oxygenase-type substrates. When the enzyme was pretreated with p-chloromercuribenzoate and then tested with the oxygenase-type substrates, there was a parallel increase in the oxidase and dehydrogenase activities concomitant with a decrease in the original oxygenase activity. These results suggested that the dehydrogenase reaction catalyzed by this enzyme was correlated with the oxidase reaction rather than with the oxygenase reaction.

Alkylamine-dependent oxidation and oxygenation of α-monoamino acids by lysine monooxygenase

Abstract Lysine monooxygenase, a pseudomonad flavoprotein, was almost inactive with an α-monoamino acid as substrate, but the addition of an alkylamine, a counterpart of the fragmented lysine molecule, caused marked oxygen consumption. The rate of oxygen consumption was examined and compared with various combinations of α-monoamino acids and alkylamines. Hydrogen peroxide was formed and the corresponding α-keto acid was produced from each amino acid. In most cases, the hydrogen peroxide formation was nearly equal to the oxygen consumption. In some other cases, however, the oxygen consumption exceeded the hydrogen peroxide formation, and the production of an acid amide in addition to the α-keto acid was qualitatively demonstrated. Thus, both an oxidative deamination and an oxygenative decarboxylation of the same substrate occurred concomitantly, although the ratio of the two types of reaction varied with different combinations of amino acids and alkylamines. Alkylamines at higher concentrations competitively inhibited the lysine oxygenation, whereas lower concentrations of alkylamines stimulated the lysine oxygenation by decreasing the sigmoidicity of the saturation curve for lysine. These results suggest a dual function of the alkylamine; i.e., the interaction with the active site as a fragment of substrate and the effect on the regulatory property of the enzyme.