Fumio Takeuchi

Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages

Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Iκ-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Iκ-Bα was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Iκ-Bα.

Purification, characterization and identification of rat liver mitochondrial kynurenine aminotransferase with α-aminiadipate aminotransferase

Abstract Kynurenine aminotransferase ( l -kynurenine:2-oxoglytarate aminotransferase (cyclizing), EC 2.6.1.7) was purified 378-fold from rat liver mitochondria by digitonin solubilization, heat treatment, DEAE-Sepharose CL-6B chromatography, Sephadex G-100 gel filtration, hydroxypatite chromatography and chromatofocusing. Elution patterns of α-aminoadipate aminotransferase (EC 2.6.1.39) activity were identical with those of kynurenine aminotransferase activity on all column chromatographies. The ratios of the two specific activities were constant throughout the purification. On polyacrylamide gel electrophoresis both activities were detected at the same position. Both enzymatic activities showed the same inactivation curves upon heat inactivation at various temperatures. α-aminoadipate showed competitive inhibition against kynurenine or 3-hydroxykynurenine, α-Ketoadipate was utilized in the kynurenine aminotransferase reaction as an amino acceptor in place of α-ketoglutarate. The K m value for α-ketoadipate was 10 μM, lower than for α-ketoglutarate. these observations indicate that kynurenine aminotransferase is identical with α-aminoadipate aminotransferase. The K m values of purified kynurenine aminotransferase were determined at pH 6.5 as: kynurenine, 4.3 mM; pyridoxal 5′-phosphate, 4.2 μM; α-ketoglutarate, 20 μM (kynurenine substrate), and 3-hydroxykynurenine, 5.7 mM; pyridoxal 5′-phosphate, 1.7 μM; α-ketoglutarate, 13 μM (3-hydroxykynurenine substrate). The enzyme was strongly inhibited by Hg 2+ and p -chloromercuribenzoate.

AMINO-ACID SEQUENCE OF RAT LIVER KYNURENINASE

Amino-acid sequence of kynureninase purified from rat liver cytosol was determined by an amino-acid sequencer. The enzyme was degraded to small peptides with cyanogen bromide, TPCK-trypsin, endoproteinase Glu-C, lysyl endoprotease and alpha-chymotrypsin. The enzyme subunit consisted of 464 amino acids, and the molecular weight of subunit was determined to be 52,510. The coenzyme pyridoxal phosphate-binding residue was lysine of which position was 276, and the N-terminal residue was N-acetylmethionine. The homology search between this enzyme and the other pyridoxal phosphate-dependent enzymes showed that kynureninase was similar to mitochondrial aspartate aminotransferase, and also to cystathionine gamma-synthase and gamma-lyase to a lesser extent.

Calcium uptake into renal brush border membranes in vitamin B6 deficient rats

The calcium uptake into renal brush border membrane vesicles, which has been purified from normal or vitamin B6 deficient rat renal cortex by calcium precipitation, was investigated. The values of Km and Vmax were determined to be 1.89 mM and 4.26 nmol of Ca2+/mg of protein per 20s in vitamin B6 deficient rats, respectively. This Vmax was lower than that of normal rats. The chemical compositions of renal brush border membranes did not display a difference in normal and vitamin B6 deficient rats. The amount of brush border membranes isolated from 1 gram of renal cortex in vitamin B6 deficient rats was less than in normal rats.