Ryo Kido’s research while affiliated with Wakayama University and other places

Antioxidant activities of caffeoyltryptophan were investigated by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging system, the superoxide anion generation system and the superoxide anion-mediated linoleic acid peroxidation system. At 10 microM, caffeoyltryptophan showed greater scavenging activity on DPPH than dl-alpha-tocopherol or ascorbic acid. DPPH radical scavenging activity of caffeoyltryptophan increased dose-dependently at concentrations ranging from 1 to 50 microM; 1 mol of caffeoyltryptophan reacted with ca 4 mol of radical. Caffeoyltryptophan caused 80% inhibition of superoxide anion generation at 50 microM. The inhibitory activity of caffeoyltryptophan was as strong as that of 5-caffeoylquinic acid. Caffeoyltryptophan inhibited the formation of conjugated diene from linoleic acid. The inhibitory activity increased in the order caffeic acid < 5-caffeoylquinic acid < caffeoyltryptophan < dl-alpha-tocopherol. Effects on the in vitro haemolysis and peroxidation of mouse erythrocytes induced by H2O2 were also examined. Caffeoyltryptophan exhibited strong inhibitory activities; Tryptophan was ineffective in these systems. These data suggest that caffeoyltryptophan may be a natural antioxidant in the human diet and, as such, may intervene in toxicological processes that are mediated by radical mechanisms.

In the mammalian brain, kynurenine aminotransferase (KAT) is pivotal to the synthesis of kynurenic acid, a preferential antagonist at the strychnine-insensitive NMDA-glycine site. As NMDA receptors are involved in autonomic function, we have examined the immunohistochemical localization of KAT in the medulla and spinal cord of the rat. KAT immunoreactivity (KAT-li) was found throughout these areas, in both glia and neurons. Unlike the mainly astrocytic localization in forebrain structures, KAT-li was predominantly neuronal, notably in areas important for blood pressure and heart rate regulation: ventral medulla, nucleus ambiguus, nucleus of the solitary tract and intramediolateral cell column of the spinal cord. The presence of KAT in these nuclei supports a neuromodulatory role for kynurenic acid in NMDA-mediated autonomic function.

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.

Two types of dioxygenases that catalyze the oxidative cleavage of the pyrrole ring of tryptophan by insertion of molecular oxygen to yield N-formylkynurenine have been reported (Feigelson and Brady, 1974). One is tryptophan 2,3-dioxygenase (TDO), and the other is indoleamine 2,3-dioxygenase (IDO). Although protoheme IX is a sole prosthetic group for both dioxygenases (Feigelson and Brady, 1974; Hirata and Hayaishi, 1975; Ishimura et al., 1980), these have proved to be distinct enzymes. TDO is a tetrameric protein (M.W. 120,000–167,000) metabolizing L-tryptophan specifically (Feigelson and Brady, 1974). This dioxygenase requires nonspecific reductants such as L-ascorbic acid for its activation in vitro (Feigelson and Brady, 1974). IDO is a monomeric protein (M.W. 41,000) (Shimizu et al., 1978) exhibiting a wide substrate specificity for various indoleamine derivatives including L-and D-tryptophan and serotonin (Ishimura et al., 1970). This enzyme can be also activated by a wide variety of reductants. In addition, methylene blue (or toluidine blue) is absolutely required as an electron mediator from a reductant to the ferric enzyme for its activation (Yamamoto and Hayaishi, 1967).

1. The present study was performed to investigate alterations in membrane characteristics of spontaneously hypertensive rats (SHR) by using an electron paramagnetic resonance (EPR) and spin-labelling methods. 2. Washed erythrocytes from SHR were examined and compared with erythrocytes from age-matched normotensive Wistar-Kyoto (WKY) rats. 3. The values of outer hyperfine splitting (2T'll) and that of the order parameter (S) obtained from EPR spectra for a spin label agent (5-nitroxide stearate) were significantly higher in the erythrocytes of SHR than in those of WKY rats. 4. When calcium (Ca2+) was loaded to erythrocytes with a Ca2+ ionophore (A 23187), the order parameter (S) of the EPR spectra showed a greater increase in SHR than in WKY rats. Furthermore, the Ca2+-induced change in the order parameter (S) of SHR was significantly antagonized by pretreatment of the Ca2+ antagonists (verapamil, diltiazem) and a calmodulin antagonist (W-7). 5. The results show that the erythrocyte membranes of SHR tolerated different spin motions from those of normotensive WKY rats in the EPR study, which might be associated with the idea that the membrane fluidity might be lower in SHR. Furthermore, the data suggest that Ca2+-calmodulin antagonists may ameliorate the Ca2+-induced changes in membrane functions in hypertension.

The physiological activating mechanism for human placental indoleamine 2,3-dioxygenase was investigated. No cell fractions of the placental homogenate were found to replace the electron-mediation activity of methylene blue from a reductant to the enzyme in the exogenous enzyme-activating system with L-ascorbic acid, B-NADPH, or B-NADH as a reductant. However, the preincubation at 37 degrees C of the supernatant fraction (10,000 x g, 30 min) prior to starting the indoleamine 2,3-dioxygenase reaction drastically caused the activation of the enzyme without added reductants and methylene blue, which suggests the existence of an endogenous indoleamine 2,3-dioxygenase-activating system. The endogenous enzyme activation was induced in the placentas delivered operatively by vacuum extraction or cesarean section much more significant degree than in those delivered normally.

Alanine-glyoxylate aminotransferase (AGT) 2 is a pyridoxal 5'-phosphate dependent, mitochondrial enzyme which, in the rat, is expressed at a high level in the kidney. The amino acid sequences of nine tryptic and seven CNBr peptides of the rat kidney AGT2 were determined. Three overlapping cDNAs encoding the AGT2 were cloned on the basis of its partial amino acid sequences by means of a polymerase chain reaction-based approach involving rat kidney poly(A)+ RNA. The complete cDNA sequence comprised 1,919 bases, and contained a 1,536-base open reading frame which encodes a polypeptide of 512 amino acid residues with a putative presequence consisting of 39 amino acid residues at the amino terminus, giving a precursor protein with a molecular mass of 57,150 Da. The sequence of AGT2 exhibits significant homology with neither peroxisomal AGT1 from human liver nor mitochondrial AGT1 from rat liver. However, the sequence of AGT2 exhibited 30.8, 29.2, and 27.1% identity with those of Escherichia coli 4-aminobutyrate aminotransferase, rat ornithine aminotransferase, and Pseudomonas cepacia 2,2-dialkylglycine decarboxylase, respectively. The active site sequences were also well conserved among these aminotransferases. AGT2, thus, is more similar to the other aminotransferases than to AGT1. The results suggest that the rat kidney AGT2 may play a biological role in amino acid metabolism distinct from that of AGT1.

Fed rats were exercised until exhaustion by almost 65% VO2max on a treadmill. In 2.5 min after the exercise, blood was collected from various vessels of the splanchnic bed. Metabolites, glucose, lactate, ketone body, and nitrogencompounds in the plasma, were measured. Glucose excretion from the liver was increased by exercise, but was not significant. The absorption by the kidney decreased to 30% by exercise. Lactate was highly absorbed by the kidney, lower limbs, and digestive tract by exercise. Exercise caused a 200-300% increase of the plasma beta-hydroxybutyrate, but the absorption by the kidney and the lower limbs was decreased. These data suggest that glucose is a good carbon source for the recovery, and that lactate is more useful than glucose, but ketone body is less effective at a very early recovery phase under fed condition. Amino acid balances in each organ except digestive tract were positive showing anabolic conditions of these organs even after exhaustive exercise at fed condition. Most amino acid concentrations in the plasma tended to decrease to 60-90% by exercise. Amino acids were excreted from the digestive tract, and were eventually absorbed by the liver in both rested and exercised rat. The digestive tract, therefore, seems to be a primary amino acids pool to supply them to the liver during the inter meal. Urea excretion from the liver was more than the absorbed ammonia showing that active deamination from amino acids was carrying on. The resulted carbon skeletons of the amino acids might be used for the gluconeogenesis in the liver.

The tryptophan pyrrole-ring cleavage enzyme (TPCE) was detected in the yeast Saccharomyces cerevisiae. TPCE activity existed constitutively and was markedly induced by culturing the cells in a medium containing 0.1% (w/v) L-tryptophan. We purified partially the enzyme from the L-tryptophan-induced cells by phospho-cellulose column chromatography. The partially purified enzyme was stimulated solely by L-ascorbic acid, a nonspecific reductant, suggesting that the yeast TPCE is not indoleamine 2,3-dioxygenase, but rather tryptophan 2,3-dioxygenase. The enzyme metabolized L-tryptophan preferentially, and D-tryptophan slightly. KCN and NaN3, exogenous ligands of heme, inhibited the enzyme activity drastically, indicating that yeast tryptophan 2,3-dioxygenase contains heme(s) in its active site. The optimal pH of the enzyme was 6.5. Upon two-dimensional polyacrylamide gel electrophoresis, a protein staining spot was identified that was induced by L-tryptophan and whose intensity changed in correlation with the tryptophan 2,3-dioxygenase activity after phospho-cellulose column chromatography. This protein, exhibiting a molecular weight of approximately 38,000 and an isoelectric point of approximately pH 8.0, may be identified as a subunit of yeast tryptophan 2,3-dioxygenase.