Involvement of D-amino acid oxidase in elimination of D-serine in mouse brain.

The physiological role of D-amino acid oxidase (EC 1. 4. 3. 3) in mouse brain is described. The presence of D-enantiomers of neutral common amino acids was surveyed in the brain. D-serine was shown to be present at high concentration only in regions where the enzyme activity was low. In normal mice whose D-amino acid oxidase activity was much higher in the cerebellum than in the cerebrum, free D-serine content was apparently lower in the cerebellum than in the cerebrum. In mice of a mutant strain lacking D-amino acid-oxidase activity, the free D-serine level was remarkably high both in the cerebrum and cerebellum. The results suggest that the enzyme is involved in the elimination of free D-serine in the cerebellum.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice.

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

D-aspartate oxidase activity and D-aspartate content in a mutant mouse strain lacking D-amino acid oxidase

A mutant mouse strain ddY/DAO- lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.     

D-Amino acid oxidase: new findings

The most recent research on D-amino acid oxidases and D-amino acid metabolism has revealed new, intriguing properties of the flavoenzyme and enlighted novel biotechnological uses of this catalyst. Concerning the in vivo function of the enzyme, new findings on the physiological role of D-amino acid oxidase point to a detoxifying function of the enzyme in metabolizing exogenous D-amino acids in animals. A novel role in modulating the level of D-serine in brain has also been proposed for the enzyme. At the molecular level, site-directed mutagenesis studies on the pig kidney D-amino acid oxidase and, more recently, on the enzyme from the yeast Rhodotorula gracilis indicated that the few conserved residues of the active site do not play a role in acid-base catalysis but rather are involved in substrate interactions. The three-dimensional structure of the enzyme was recently determined from two different sources: at 2.5-3.0 A resolution for DAAO from pig kidney and at 1.2-1.8 A resolution for R. gracilis. The active site can be clearly depicted: the striking absence of essential residues acting in acid-base catalysis and the mode of substrate orientation into the active site, taken together with the results of free-energy correlation studies, clearly support a hydrid transfer type of mechanism in which the orbital steering between the substrate and the isoalloxazine atoms plays a crucial role during catalysis.     

D-Aspartate oxidase activity and D-aspartate content in a mutant mouse strain lacking D-amino acid oxidase

Summary A mutant mouse strain ddY/DAO^– lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.     

The presence of free D-serine,D-alanine and D-proline in human plasma

Twelve neutral free amino acids, i. e., serine, threonine, glutamine, asparagine, alanine, proline, methionine, tyrosine, valine, leucine, isoleucine and phenylalanine, were surveyed for the presence of D-enantiomers in plasma samples from patients with renal diseases and from normal subjects. D-serine, D-alanine and D-proline were found in the patient's plasma. The highest concentrations (D/L ratio) of D-serine, D-alanine and D-proline were 0.2362, 0.2087 and 0.0986, respectively. The sum of the contents of the three D-amino acids in a plasma sample correlated with the serum creatinine level of the subject. No D-amino acid was shown to be present in the plasma proteins.     

The presence of free D-serine, D-alanine and D-proline in human plasma.

Twelve neutral free amino acids, i.e. serine, threonine, glutamine, asparagine, alanine, proline, methionine, tyrosine, valine, leucine, isoleucine and phenylalanine, were surveyed for the presence of D-enantiomers in plasma samples from patients with renal diseases and from normal subjects. D-serine, D-alanine and D-proline were found in the patient's plasma. The highest concentrations (D/L ratio) of D-serine, D-alanine and D-proline were 0.2362, 0.2087 and 0.0986, respectively. The sum of the contents of the three D-amino acid was shown to be present in the plasma proteins.     

Physiological functions of D-amino acid oxidases: from yeast to humans

D-Amino acid oxidase (DAAO) is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-isomers of neutral and polar amino acids. This enzymatic activity has been identified in most eukaryotic organisms, the only exception being plants. In the various organisms in which it does occur, DAAO fulfills distinct physiological functions: from a catabolic role in yeast cells, which allows them to grow on D-amino acids as carbon and energy sources, to a regulatory role in the human brain, where it controls the levels of the neuromodulator D-serine. Since 1935, DAAO has been the object of an astonishing number of investigations and has become a model for the dehydrogenase-oxidase class of flavoproteins. Structural and functional studies have suggested that specific physiological functions are implemented through the use of different structural elements that control access to the active site and substrate/product exchange. Current research is attempting to delineate the regulation of DAAO functions in the contest of complex biochemical and physiological networks.     

Serotonin metabolism in the CNS in cerebellar ataxic mice

The metabolism of 5-hydroxytryptamine (5-HT) in the CNS was investigated in four kinds of morphologically different ataxic mice; reeler, staggerer, weaver and Purkinje cell degeneration mutants, and in hypocerebellar mice experimentally produced by injection of cytosine arabinoside. 5-HT and 5-hydroxyidoleacetic acid concentrations and tryptophan hydroxylase (TrpOH) activity were measured in the cerebrum, cerebellum and brain stem, respectively. TrpOH activity was significantly reduced only in the reeler mouse. The enhancements of the cerebellar 5-HT metabolism observed in the ataxic mice other than the reeler were supposed to be pseudo-enhancements subsequent to the cerebellar hypoplasia.     

D-amino acids in mouse tissues are not of microbial origin

Neutral free D-amino acid contents in the serum, kidney, liver, brain, small intestine and urine in germ-free mice and those in specific pathogen-free mice were compared. No significant difference was found. This strongly suggests that the free D-amino acids which were shown to be present in mice in our previous work did not originate from the enteric microbial flora.     

D-Amino acids in mouse tissues are not of microbial origin

Summary Neutral free D-amino acid contents in the serum, kidney, liver, brain, small intestine and urine in germ-free mice and those in specific pathogen-free mice were compared. No significant difference was found. This strongly suggests that the free D-amino acids which were shown to be present in mice in our previous work^{1, 2} did not originate from the enteric microbial flora.     

Immunochemical studies on Rhodotorula gracilis D-amino acid oxidase.

Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified from R. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.     

Immunochemical studies onRhodotorula gracilis D-amino acid oxidase

Summary Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified fromR. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.     

The presence of free D-amino acids in mouse tissues

Summary The presence of free D-amino acids in mouse kidney, liver, brain, heart, lung, thymus and serum has been shown with an enzymic microdetermination method. The D-amino acid levels were higher in the extracts of kidney and liver than in those from other organs.     

The presence of free D-amino acids in mouse tissues

The presence of free D-amino acids in mouse kidney, liver, brain, heart, lung, thymus and serum has been shown with an enzymic microdetermination method. The D-amino acid levels were higher in the extracts of kidney and liver than in those from other organs.     

Ultrastructural localization of catalase and D-amino acid oxidase in 'normal' fetal mouse liver

In the hepatocytes of 'normal' fetal mice from mothers which were carriers of muscular dysgenesis, catalase and D-amino acid oxidase (DAAO) positive as well as negative peroxisomes were observed. DAAO reaction product was occasionally localized in patches around cell membranes and DAAO-positive peroxisomes were frequently observed near mitochondria.     

Neurochemical correlates of alloxan diabetes: Protein and ribonucleic acid levels in the different regions of the rat brain

Summary The levels of protein and ribonucleic acid in the cerebrum, cerebellum, optic lobes and medulla oblongata of normal and alloxan-diabetic rats were measured. In general, the protein content and levels of ribonucleic acid in the broad compartments of the brain of rat decreased during diabetes.Acknowledgment. The authors thank Dr.A. R. Kasturi Bai, Head of the Department of Zoology, Bangalore University, for providing facilities during the study.     

Autoradiographic localization of the uptake of 3H-beta-alanine in rat nervous tissue cultures.

Autoradiographic studies on the uptake of 3H-beta-alanine have shown that, in spinal cord and brain stem cultures, both neurones and glial cells have accumulated the amino acid. In contrast, in cultures of cerebellum and dorsal root ganglia, 3H-beta-alanine was only t aken up by glial elements.     

Cerebral edema in the rat with galactosamine induced severe hepatitis

Summary With D-galactosamine hydrochloride severe hepatitis was induced in rats and the water content of cerebrum, cerebellum and brain stem determined. The animals showed a parallel in cerebral water content and occurrence of cerebral symptoms.