Yukio Shibata

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.

Acclimatization to hypoxia modulates the tryptophan 2,3-dioxygenase activity in rats exposed to simulated high altitude

1. Exposure of rats to an 8000 m altitude increased the hepatic tryptophan 2,3-dioxygenase (EC 1.13.1.12) activity. 2. Acclimatization to hypoxia by a repeated exposure to an altitude of 5000 m induced a marked decrease in liver tryptophan dioxygenase activity after the rats were subjected to an 8000 m altitude, but a pre-exposure to 4000 m altitude showed no effect on the enzyme activity. 3. Plasma tryptophan was rapidly decreased by exposure to 8000 m altitude to the same extent in the acclimatized and non-acclimatized rats. 4. Plasma tryptophan may be utilized as the substrate for tryptophan dioxygenase in liver of the non-acclimatized rats under highly hypoxic conditions; however, acclimatized rats can reserve tryptophan as the substrate for the alternative metabolism other than the degradation pathway in liver.

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.

Xanthurenic Acid and Zinc

Prof. H. Tornita of the Department of Otorhinolaryngology, Nihon University, has been carrying out experiments to determine the relationship between Zn2+ and taste abnormality in diabetes mellitus. Normal taste capacity was recovered by the administration of ZnSO4 to these patients. K. Okamoto of Kinki University [1] discovered oxine (8-hydroxy quinoline) diabetes; its mechanism might be due to chelation with Zn2+.

Reactivities of human urinary prokallikrein and kallikrein toward rabbit antibodies with special reference to enzyme immunoassay

Human urinary prokallikrein and kallikrein have been analyzed by means of solid phase enzyme immunoassay (EIA) using rabbit antibodies. The anti-kallikrein antibody-immobilized EIA detected both kallikrein and prokallikrein. However, only kallikrein was detectable at concentrations below 20 micrograms/1. The anti-prokallikrein antibody-immobilized EIA detected both kallikrein and prokallikrein, but the binding maximum for kallikrein was about one-third less than that for prokallikrein. Similar difference in reactivity of kallikrein and prokallikrein toward each antibody was observed with immunoaffinity column chromatography and single radial immunodiffusion. The results show that immunochemical properties of human urinary prokallikrein and kallikrein differ distinctly, and conflicting results on detectability of kallikrein and prokallikrein in human urine with EIA or radioimmunoassay using the anti-kallikrein antibody was due to the difference in reactivity of the two forms of kallikrein toward the immobilized antibody.