Biomedical subjects

E Okuno

Publications and source records attributed to E Okuno.

At least 55 records · Page 3Linked to original sources

PubMed1989

Organ distribution, purification and characterization of kynureninase in Suncus murinus (Insectivora) and anthranilic acid level in the serum.

1. The highest kynureninase activity was measured in Suncus liver compared with other organs tested. The holo-enzyme activity detected in liver was 25%. 2. The enzyme mainly localized in liver cytosol was purified to a single protein band by heat treatment, ammonium sulfate fractionation, DEAE-sepharose, hydroxyapatite and phenyl-sepharose column chromatography. 3. The purified enzyme was effective for both kynurenine and 3-hydroxykynurenine, and showed the optimum pH at 8.5 against both substrates. 4. Km values were 250 microM, 18 microM and 70 microM for kynurenine, 3-hydroxykynurenine and pyridoxal 5'-phosphate, respectively. Various compounds such as histidine, aspartate and nicotinamide enhanced the enzyme activities. 5. Anthranilate, the product of kynureninase, was present at a concentration in Suncus serum of 1.96 microM. This value was higher than those of rat and human.

Animals↗

PubMed1988

Immunohistochemical identification of quinolinic acid phosphoribosyltransferase in glial cultures from rat brain.

Glial cell cultures were shown to contain 3 identifiable classes of cells which could be specifically stained with antibodies directed against quinolinic acid phosphoribosyltransferase (QPRT), the catabolic enzyme of the endogenous excitotoxin quinolinic acid. Some, but not all, QPRT-positive cells also contained glial fibrillary acidic protein. These cultures may constitute an in vitro system in which cerebral quinolinic acid metabolism and function can be examined.

Animals↗

PubMed1988

Enzymatic studies on tryptophan metabolism disorder in rats chronically exposed to carbon disulfide.

Eight-week-old female Wistar rats were exposed to carbon disulfide for 6 hr a day, 5 days a week, for 12 weeks in inhalation chambers. Then the activities of the main enzymes of tryptophan metabolism (i.e., L-tryptophan 2,3-dioxygenase, indoleamine 2,3-dioxygenase, kynurenine 3-hydroxylase, kynureninase, and kynurenine aminotransferases) in their tissues were determined. The results showed that exposure to carbon disulfide caused a significant increase in the activities of kynureninase and kynurenine-2-oxoglutarate aminotransferase in the kidneys, but only a slight increase in their activities in the liver. The activities of L-tryptophan 2,3-dioxygenase and kynurenine 3-hydroxylase also tended to increase, but the increases were not statistically significant. These results suggests that the kynurenine pathway of tryptophan metabolism in the kidneys of rats exposed to carbon disulfide is activated and that the increased activities of kynurenine-2-oxoglutarate aminotransferase in the kidneys may cause the increased excretion of tryptophan metabolites after tryptophan loading as shown in an earlier study (A. Okayama, L. Fun, A. Yamatodani, Y. Ogawa, H. Wada, and S. Goto, 1987, Arch. Toxicol. 60, 460-463.

Animals↗

PubMed1988

Alanine:glyoxylate aminotransferase activities in liver of Suncus murinus (insectivora).

1. The distribution of L-alanine:glyoxylate aminotransferase (AGT) activities were found in Suncus liver, 55% in particulate fraction and 45% in supernatant. 2. 65% of AGT activities in particulate were dependent on AGT isoenzyme 2 (AGT 2) having molecular weight 210,000, the remainder (35%) of AGT activities were dependent on AGT isoenzyme 1 (AGT 1) which have aminotransferase activity for serine. AGT activities in supernatant were dependent on AGT 1, AGT 2 and alanine:2-oxoglutarate aminotransferase (GPT), and their activity ratios were 10, 15 and 75%, respectively. 3. Km values for alanine were 0.52 mM; AGT 1, 3.3 mM; AGT 2, 0.88 mM; GPT measuring with AGT activity. AGT activity of GPT was inhibited by addition of glutamate and its Ki value was 1.8 mM. 4. Some other properties of AGT 1, AGT 2 and GPT are described.

Alanine Transaminase↗

PubMed1988

Localization of quinolinic acid metabolizing enzymes in the rat brain. Immunohistochemical studies using antibodies to 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase.

Specific antibodies raised in rabbits against 3-hydroxyanthranilic acid oxygenase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) were used in immunohistochemical studies to map the cellular localization of the quinolinic acid metabolizing enzymes in the adult male rat brain. 3-Hydroxyanthranilic acid oxygenase immunoreactivity was found to be present in glial cells of presumed astroglial identity, as judged by co-localization with glial fibrillary acidic protein. 3-Hydroxyanthranilic acid oxygenase-immunoreactive glial cells were present in all brain regions and within major fiber tracts. The density of 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells as well as the intensity of staining of these cells differed among brain regions. In general, telencephalic acid diencephalic areas harbored a larger number of 3-hydroxyanthranilic acid oxygenase-positive cells than did mesencephalic regions. In the former regions the caudate nucleus, septum, nucleus accumbens, neocortex and hippocampus were particularly enriched in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In the thalamus, regional differences were noted with regard to the intensity of staining among glial cells with high densities of 3-hydroxyanthranilic acid oxygenase cells in the anteroventral, reticular and ventromedial nuclei. In the inferior and superior colliculi, numerous 3-hydroxyanthranilic acid oxygenase-positive glial cells were found in all layers. In the hypothalamus, 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells were encountered in the zona incerta, the lateral hypothalamic area, the caudal preoptic region and in the dorsomedial nucleus. In the mesencephalon, the substantia nigra contained numerous, moderately stained cells. At caudal levels of the brain-stem, a relatively large number of cells was detected in the nucleus of the solitary tract, the pontine nucleus and in the fascial nerve nucleus, while other nuclei, such as the reticular formation and the area postrema were relatively poor in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In addition to staining of glial cells, neuronal cell bodies containing 3-hydroxyanthranilic acid oxygenase immunoreactivity were detected in the main and in the accessory olfactory bulb, as well as in the ventromedial nucleus of the hypothalamus. Quinolinic acid phosphoribosyltransferase immunoreactivity was observed within glial cells and in association with neuronal cell bodies. Some, but not all, quinolinic acid phosphoribosyltransferase positive glial cells contained glial fibrillary acidic protein (Köhl

3-Hydroxyanthranilate 3,4-Dioxygenase↗

PubMed1988

3-Hydroxyanthranilate oxygenase activity is increased in the brains of Huntington disease victims.

An excess of the tryptophan metabolite quinolinic acid in the brain has been hypothetically related to the pathogenesis of Huntington disease. Quinolinate's immediate biosynthetic enzyme, 3-hydroxyanthranilate oxygenase (EC 1.13.11.6), has now been detected in human brain tissue. The activity of 3-hydroxyanthranilate oxygenase is increased in Huntington disease brains as compared to control brains. The increment is particularly pronounced in the striatum, which is known to exhibit the most prominent nerve-cell loss in Huntington disease. Thus, the Huntington disease brain has a disproportionately high capability to produce the endogenous "excitotoxin" quinolinic acid. This finding may be of relevance for clinical, neuropathologic, and biochemical features associated with Huntington disease.

3-Hydroxyanthranilate 3,4-Dioxygenase↗

PubMed1988

Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain.

Quinolinic acid phosphoribosyltransferase (QPRT) [EC 2.4.2.19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physiocochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170,000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 microM for the substrate (quinolinic acid) and 23 microM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1 mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.

Animals↗

PubMed1988

Organ distribution of rat kynureninase and changes of its activity during development.

Kynureninase activity was measured in various organs of the rat by a sensitive assay method based on the use of an HPLC system. High activities were detected in liver, kidney and spleen, and much lower activities in adrenals, intestine, lung, heart, brain, skeletal muscle and pancreas. Kynureninase of liver, kidney and spleen showed the same molecular weight (110,000) and the same isoelectric point (pI 5.4). They also showed the same properties of heat stability and apparent optimum pH. The enzymes in kidney and spleen were localized in the cytosol. The developmental study showed increasing activities of the enzyme after birth and a maximum on the 60th day in both liver and spleen. The activity in kidney increased after birth and reached a plateau on the 30th day.

Aging↗

PubMed1988

Quinolinic acid metabolism in the rat brain. Immunohistochemical identification of 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase in the hippocampal region.

Quinolinic acid (QUIN) is a potent endogenous excitotoxin, which has been shown to be present in the brain (Wolfensberger et al., 1983). In order to study the cellular localization of QUIN metabolism in the hippocampus, specific antibodies raised against purified rat liver 3-hydroxyanthranilic acid oxygenase (3HAO) and quinolinic acid phosphoribosyltransferase (QPRT), the enzymes directly responsible for QUIN synthesis and catabolism, respectively, were used for immunohistochemical studies in the adult male rat. Cells containing 3HAO immunoreactivity (3HAO-i) were present in all subfields of the hippocampal region, including the area dentata, Ammon's horn, the subicular complex, and the entorhinal area. The highest density of 3HAO-i cells was found in the molecular layer of Ammon's horn and in the hilus of area dentata, while the granular cell layer of area dentata and stratum pyramidale of Ammon's horn contained the lowest number of 3HAO-stained cells. A majority of hippocampal 3HAO-i cells were also stained with monoclonal antibodies against glial fibrillary acidic protein (GFAP) or S-100 protein, suggesting that 3HAO-i is present primarily in astrocytes. At the ultrastructural level, 3HAO-i was found to be distributed uniformly throughout the cytoplasm, with intense immunostaining present in the internal and the external layers of the mitochondria. QPRT-i was detected in 3 morphologically distinct cell types present in all parts of the hippocampus. The total number of QPRT-i cells was lower than that of the 3HAO-i cells. QPRT-i cells were relatively numerous in the molecular and radial layers of Ammon's horn, while they occurred only sporadically in stratum pyramidale of Ammon's horn and in the granular cell layer of area dentata. Many QPRT-i cells stained with antibodies against GFAP and S-100, but the proportion of cells in which QPRT was colocalized with these glial marker proteins was lower than that for 3-HAO-i cells. At the ultrastructural level, 2 types of QPRT-i glial cells were detected. The smaller cell type had a diffuse cytoplasmic staining, while the larger cell type, which also contained glial filaments, showed diffuse cytoplasmic staining and intense staining of lysosomal structures. The observation that 3HAO and QPRT only partially coexist in hippocampal glial cells suggests that while synthesis and catabolism of QUIN may occur in the same glial cells, catabolism of QUIN can also take place in cells lacking the synthetic enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Increased quinolinic acid metabolism following neuronal degeneration in the rat hippocampus.

The excitotoxic brain metabolite quinolinic acid (QUIN) has been hypothetically linked to the pathogenesis of seizure disorders and other neurodegenerative events affecting the hippocampal formation. Its biosynthetic enzyme, 3-hydroxyanthranilic acid oxygenase (3-HAO) and its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRT), can be used as markers for the cellular localization of the brain's QUIN system. Measured between 2 days and 2 months following intrahippocampal ibotenic acid injections, the activities of both enzymes increased at the lesion site due to the synthesis of new enzyme protein. The time course of the increase in 3-HAO activity coincided with that of the known astrocytic proliferation following excitotoxic insults. It is less obvious if the elevation in QPRT activity, too, is related to an increase in the number of reactive glial cells. No changes in the activity of hippocampal 3-HAO or QPRT were noted 7 or 60 days after cholinergic deafferentation by fornix-fimbria transection nor were any changes observed in the contralateral hippocampus at any time-point following the ibotenate lesion. These data raise the possibility that a feed-forward mechanism, resulting in ever increasing amounts of QUIN in the brain, may be operant in situations of progressive hippocampal nerve cell loss.

Animals↗

PubMed1987

Quinolinic acid phosphoribosyltransferase: preferential glial localization in the rat brain visualized by immunocytochemistry.

The excitotoxic brain metabolite quinolinic acid has been hypothetically linked to the pathogenesis of neurodegenerative disorders. By using antibodies prepared against a homogeneous preparation of its catabolic enzyme, quinolinic acid phosphoribosyltransferase [QPRTase; nicotinate-nucleotide:pyrophosphate phosphoribosyltransferase (carboxylating), EC 2.4.2.19], immunocytochemical methods were applied to assess the cellular and subcellular localization of quinolinic acid in the rat brain. On the light-microscopic level, the enzyme was found to be preferentially associated with glial elements of variable morphology. In addition to its presence in glial cells, QPRTase was contained in tanycytes and ependymal cells of the cerebral ventricles and, sporadically, in neurons. Overall, QPRTase immunoreactivity was noted in every brain region studied, the histological pattern being in good accordance with the regional variation of enzyme activity established in biochemical studies. As judged on the ultrastructural level, QPRTase, in all cell types examined so far, was often noted in densely stained roundish cytoplasmic bodies (0.1-0.8 micron in diameter), which were bounded by a single membrane. In functional terms, these structures may represent early lysosomes, secretory granules, or residual bodies. The particular anatomical arrangement of the quinolinic acid system may reflect the brain's defense strategy against detrimental effects of the endogenous excitotoxin.

Animals↗

PubMed1987

Rat 3-hydroxyanthranilic acid oxygenase: purification from the liver and immunocytochemical localization in the brain.

3-Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6), the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37-38,000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical Km of 3 microM for the substrate 3-hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe2+) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double-diffusion experiments. Immunohistochemical studies using purified anti-rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brain's quinolinic acid system.

3-Hydroxyanthranilate 3,4-Dioxygenase↗

PubMed1986

Quinolinic acid: a pathogen in seizure disorders?

The evidence for an involvement of QUIN in human seizure disorders is clearly circumstantial. Importantly, QUIN is not a classical neurotransmitter and may thus play only a negligible or no role at all in normal brain function (Foster et al., 1984). We have yet to understand if and how such a possibly inert metabolite may turn into a pathogen. Several crucial questions remain to be addressed before a case can be made for a 'quinolinic acid hypothesis' of temporal lobe epilepsy. Among the most prominent ones figure the extracellular concentration of QUIN in the human brain under normal and pathological ('epileptic') conditions, the relationship between QUIN metabolism in the brain and its extracellular concentration and, a related issue, the regulation of cerebral QUIN metabolism (i.e., turnover). It is of equal importance to assess if NMDA-receptors, particularly those in the hippocampus and other parts of the limbic system, can exert a modulatory function upon brain QUIN. Unquestionably, future experiments with selective NMDA-antagonists will prove useful for the elucidation of such possible (feedback) interactions.

Afferent Pathways↗

PubMed1986

A radioenzymatic assay for quinolinic acid.

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.

Adult↗

PubMed1985

Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain.

Quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) was purified 3600-fold from rat liver and 280-fold from rat brain. Kinetic analyses (Km = 12 microM for the substrate quinolinic acid and Km = 23 microM for the cosubstrate phosphoribosylpyrophosphate), physicochemical properties of the purified enzymes, inhibition by phthalic acid (Ki = 1.4 microM) and molecular weight determination (Mr 160 000 for the holoenzyme, consisting of five identical 32 kDa subunits) indicated the structural identity of quinolinic acid phosphoribosyltransferase from the two rat tissues. This was further confirmed immunologically, using antibodies raised against purified rat liver quinolinic acid phosphoribosyltransferase. Rat quinolinic acid phosphoribosyltransferase differs in several aspects from quinolinic acid phosphoribosyltransferase isolated from other organisms. The purified enzyme will prove a useful tool in the examination of a possible role of quinolinic acid in cellular function and/or dysfunction.

Animals↗

PubMed1985

Peroxisome localized human hepatic alanine-glyoxylate aminotransferase and its application to clinical diagnosis.

The subcellular localization of alanine-glyoxylate aminotransferase (EC 2.6.1.44 L-Alanine: glyoxylate aminotransferase) of adult human liver was examined by sucrose density gradient centrifugation. The enzyme sedimented at the same density as catalase, indicating that it was localized in the peroxisomes. Alanine-glyoxylate aminotransferase activity in the liver of patients with cirrhosis was about 65% of that of normal liver or 71% of that from patients with chronic hepatitis, but its activity in the serum of patients with cirrhosis was higher than that from patients with chronic hepatitis. Patterns of activity of alanine-glyoxylate aminotransferase in liver and serum differed from those of aspartate-2-oxoglutarate aminotransferase and ornithine carbamoyltransferase that have a different intracellular location. Serum immunoreactive alanine-glyoxylate aminotransferase (Im-AGT) was measured by enzyme-linked immunoadsorbent assay (ELISA). The Im-AGT levels (mean +/- SEM) in acute (80 +/- 13 micrograms/L) and chronic (72 +/- 4 micrograms/L) hepatitis were higher than those of normal controls (44 +/- 1 micrograms/L). However, the difference between acute and chronic hepatitis was not statistically significant. The level in liver cirrhosis (54 +/- 3 micrograms/L) was lower than those of the hepatitides but higher than that of normal controls. The apparent half-life of serum Im-AGT of patients who underwent liver lobectomy by a microwave tissue coagulation method was approximately 3-4 days.

Adult↗

PubMed1984

Intracellular localization and characterization of 3-hydroxykynureninase in human liver.

3-hydroxykynureninase in human liver was present in cytosol and mitochondria. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent Km values of the enzyme were 7.7 X 10(-5) M for 3-hydroxykynurenine, 1.0 X 10(-3) M for kynurenine and 2.5 X 10(-6) M for pyridoxal 5'-phosphate with 3-hydroxykynurenine. Some other properties of purified enzymes are described.

Aged↗

PubMed1984

Characteristics of pancreatic branched chain amino acid aminotransferases from rat, man and dog.

Branched chain amino acid aminotransferase (EC 2.6.1.42) from pancreas of rat, man and dog was highly purified and characterized. Rat pancreas contained only one type of branched chain amino acid aminotransferase, but human and dog pancreas contained two types. These were named Enzyme I and Enzyme III according to the classification by Aki et al. (1969). Five enzymes purified were activated by the addition of 2-mercaptoethanol and glutathione. Enzyme III has lower Km values for amino acids and 2-oxo acids than Enzyme I. Some other properties of purified enzymes are described.

Animals↗