D-aspartate Oxidase Research Articles

D-Aspartate oxidase and D-amino acid oxidase are localised in the peroxisomes of terrestrial gastropods

D-Aspartate oxidase and D-amino acid oxidase were found in high activity in the tissues of representative species of terrestrial gastropods. Analytical subcellular fractionation demonstrated that both of these oxidases co-localised with the peroxisome markers, acyl-CoA oxidase and catalase, in the digestive gland homogenate. Electron microscopy of peak peroxisome fractions showed particles of uniform size with generally well preserved variably electron-dense matrices bounded by an apparently single limiting membrane. Many of the particles exhibited a core region of enhanced electron density. Catalase cytochemistry of peak fractions confirmed the peroxisome identity of the organelles. Peroxisome-enriched subcellular fractions were used to investigate the properties of gastropod D-aspartate oxidase and D-amino acid oxidase activities. The substrate and inhibitor specificities of the two activities demonstrated that two distinct enzymes were present analogous to, but not identical to, the equivalent mammalian peroxisomal enzymes.

Microbial oxidases of acidic d-amino acids

Two microbial oxidases of acidic d-amino acids have been purified to homogeneity. One is a d-aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with d-aspartate and is far more active toward d-aspartate than d-glutamate. The other is a d-glutamate oxidase of Candida boidinii 2201 that preferred d-glutamate to d-aspartate as a substrate in terms of k cat/ K m, but was not induced very effectively by d-glutamate. The most potent competitive inhibitor of the C. humicolus d-aspartate oxidase was malonate, and that of the C. boidinii d-glutamate oxidase was d-malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus d-aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus d-aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K d for the apoprotein-FAD complex was very low (8.2×10 −12 M) consistent with the observed tight binding. The C. humicolus d-aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral d-amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.

D-Aspartate oxidase and free acidic d-amino acids in lower vertebrates

d-Aspartate oxidase and free d-aspartate and/or d-glutamate have been found in various tissues of lower vertebrates including avians, amphibians and fishes. The enzymes of those animals were similar to the mammalian enzymes in substrate and inhibitor specificity. N-Methyl- d-aspartate is the best substrate of the enzymes of those animals, followed by d-aspartate, meso-2,3-diaminosuccinate and d-glutamate. In avians and amphibians, the highest activity was found in the kidneys, whereas, in fishes, hepatic activity was equal to or greater than renal activity. Male chickens had significantly higher activities in the liver, kidneys and pancreas than female. However, no significant sex difference was observed in other animals. Substantial amounts of d-aspartate and/or d-glutamate were detected in all tissues examined, irrespective of species. With the exception of the spleen or testes of some species, the d-glutamate content was equal to or greater than the d-aspartate content. The amounts of d-enantiomer present were significantly different between two sexes in several tissues of avians and amphibians. However, no common relationship was observed between the d-enantiomer contents and d-aspartate oxidase activity. The involvement of d-aspartate in the control of androgen secretion by the ovary was demonstrated in a green frog.

The role of D-aspartic acid and N-methyl-D-aspartic acid in the regulation of prolactin release.

In this study, using an enzymatic HPLC method in combination with D-aspartate oxidase, we show that N-methyl-D-aspartate (NMDA) is present at nanomolar levels in rat nervous system and endocrine glands as a natural compound, and it is biosynthesized in vivo and in vitro. D-aspartate (D-Asp) is its natural precursor and also occurs as an endogenous compound. Among the endocrine glands, the highest quantities of D-Asp (78 +/- 12 nmol/g) and NMDA (8.4 +/- 1.2 nmol/g) occur in the adenohypophysis, whereas the hypothalamus represents the area of the nervous system where these amino acids are most abundant (55 +/- 9 and 5.6 +/- 1.1 nmol/g for D-Asp and NMDA, respectively). When D-Asp is administered to rats by ip injection, there is a significant uptake of D-Asp into the adenohypophysis and a significant increase in the concentration of NMDA in the adenohypophysis, hypothalamus and hippocampus, suggesting that D-Asp is an endogenous precursor for NMDA biosynthesis. Experiments conducted on tissue homogenates confirm that D-Asp is the precursor of the NMDA and that the enzyme catalyzing this reaction is a methyltransferase. S-adenosyl-L-methionine (SAM) is the methyl group donor. In vivo experiments consisting of ip injections of sodium D-aspartate show that this amino acid induced a significant serum PRL elevation and this effect is dose and time dependent. In vitro experiments conducted on isolated adenohypophysis or adenohypophysis coincubated with the hypothalamus, showed that the release of PRL is caused by a direct action of D-Asp on the pituitary gland and also mediated by the indirect action of NMDA on the hypothalamus. Then, the latter induces the release of a putative factor that in turn stimulates the adenohypophysis reinforcing the PRL release. In conclusion, our data suggest that D-Asp and NMDA are present endogenously in the rat and are involved in the modulation of PRL release.

Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release.

Using two specific and sensitive fluorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of D-Asp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations approximately 100-fold lower than D-Asp. D-AP5, a specific NMDA receptor antagonist, inhibited D-Asp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase.

The human L-pipecolic acid oxidase is similar to bacterial monomeric sarcosine oxidases rather than D-amino acid oxidases.

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, the authors cloned the human gene to order to further study its functions. BLAST search of the translated sequence showed greatest homology to Bacillus sp. NS-129 monomeric sarcosine oxidase. The purified enzyme could use either L-pipecolic acid or sarcosine as a substrate. No homology was found to the peroxisomal D-amino acid oxidases. A further comparison of L-pipecolic acid oxidase to the two D-amino acid oxidases in peroxisomes showed that the proteins differed in many ways. First, both D-amino acid oxidase and L-pipecolic acid oxidase showed no enzyme activity in liver from Zellweger syndrome patients; D-aspartate oxidase activity was unchanged from control levels. Although all were targeted to peroxisomes, their targeting signals differed. No L-pipecolic acid oxidase was found in brain or other tissues outside of liver and kidney. The D-amino acid oxidases were similarly and more widely distributed. Finally, although D-amino acid degradation is limited to peroxisomes in mammals, L-pipecolic acid can be oxidized in either mitochondria or peroxisomes, or both.

D-aspartate oxidase is present in ovaries, eggs and embryos but not in testis of Xenopus laevis

d-aspartate oxidase (DASPO) is an FAD dependent flavoprotein which catalyzes the oxidative deamination of d-aspartate using oxygen as electron acceptor. d-aspartate and DASPO are supposed to be involved in the regulation of the central nervous system and in the animal development. This manuscript describes for the first time the presence of DASPO in Xenopus laevis fertilized eggs and embryos and suggests a different tissue distribution of this enzyme in adult male and female animals. In particular, by means of 2D-electrophoresis and affinity purified specific anti-DASPO antibodies, the enzyme was localized in fertilized eggs of X. laevis and in ovaries of adult animals but it was shown to be absent in the testis suggesting a gender specific expression. The protein from Xenopus ovaries has been purified by means of immunoprecipitation and it has M r of 30 kDa and pI of 8.1.

Occurrence of d-amino acids in a few archaea and dehydrogenase activities in hyperthermophile Pyrobaculum islandicum

The contents of d-enantiomers of serine, alanine, proline, glutamate (glutamine) and aspartate (asparagine) were examined in the membrane fractions, soluble proteins and free amino acids from some species of archaea, Pyrobaculum islandicum, Methanosarcina barkeri and Halobacterium salinarium. Around 2% ( d/ d+ l) of d-aspartate was found in the membrane fractions. In the soluble proteins, the d-amino acid content was higher in P. islandicum than that in the other archaeal cells: the concentrations in P. islandicum were 3 and 4% for d-serine and d-aspartate, respectively. High concentrations of free d-amino acids were found in P. islandicum and H. salinarium; the concentrations of d-serine (12–13%), d-aspartate (4–7%) and d-proline (3–4%) were higher than those of d-alanine and d-glutamate. This result showed a resemblance between these archaea and not bacterial, but eukaryotic cells. The presence of d-amino acids was confirmed by their digestion with d-amino acid oxidase and d-aspartate oxidase. The occurrence of d-amino acids was also confirmed by the presence of activities catalyzing catabolism of d-amino acids in the P. islandicum homogenate, as measured by 2-oxo acid formation. The catalytic activities oxidizing d-alanine, d-aspartate and d-serine at 90°C were considerably high. Under anaerobic conditions, dehydrogenase activities of the homogenate were 69, 84 and 30% of the above oxidase activities toward d-alanine, d-aspartate and d-serine, respectively. Comparable or higher dehydrogenase activities were also detected with these d-amino acids as substrate by the reduction of 2,6-dichlorophenolindophenol. No d-amino acid oxidase activity was detected in the homogenates of M. barkeri and H. salinarium.

Determination of N-methyl- d-aspartate in tissues of bivalves by high-performance liquid chromatography

The natural occurrence of N-methyl- d-aspartate (NMDA) is limited to the foot muscle of Scapharca broughtonii; it is a well known compound for its neuroexitatory activity. This paper describes a high-performance liquid chromatographic (HPLC) method for the determination of NMDA in biological extracts. The method involves removal of neutral and basic substances by anion-exchange chromatography and removal of acidic primary amino acids by treatment with o-phthalaldehyde before derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, followed by HPLC with isocratic elution with a selected mobile phase that separates the two diastereomers formed. The identity of the detected NMDA has been confirmed by a procedure using (−)-1-(9-fluorenyl)ethyl chloroformate as a derivatizing agent. The identification has been further supported by the disappearance of the peak of the NMDA derivative by pretreatment of the sample with d-aspartate oxidase. Application of the method has shown the presence of NMDA in several tissues of S. broughtonii and Scapharca subcrenata.

Purification of beef kidney d-aspartate oxidase overexpressed in Escherichia coli and characterization of its redox potentials and oxidative activity towards agonists and antagonists of excitatory amino acid receptors

The flavoenzyme d-aspartate oxidase from beef kidney (DASPO, EC 1.4.3.1) has been overexpressed in Escherichia coli. A purification procedure, faster than the one used for the enzyme from the natural source (bDASPO), has been set up yielding about 2 mg of pure recombinant protein (rDASPO) per each gram of wet E. coli paste. rDASPO has been shown to possess the same general biochemical properties of bDASPO, except that the former contains only FAD, while the latter is a mixture of two forms, one active containing FAD and one inactive containing 6-OH-FAD (9–20% depending on the preparation). This results in a slightly higher specific activity (about 15%) for rDASPO compared to bDASPO and in facilitated procedures for apoprotein preparation and reconstitution. Redox potentials of −97 mV and −157 mV were determined for free and l-(+)-tartrate complexed DASPO, respectively, in 0.1 M KPi, pH 7.0, 25°C. The large positive shift in the redox potential of the coenzyme compared to free FAD (−207 mV) is in agreement with similar results obtained with other flavooxidases. rDASPO has been used to assess a possible oxidative activity of the enzyme towards a number of compounds used as agonists or antagonists of neurotransmitters, including d-aspartatic acid, d-glutamic acid, N-methyl- d-aspartic acid, d,l-cysteic acid, d-homocysteic acid, d,l-2-amino-3-phosphonopropanoic acid, d-α-aminoadipic acid, d-aspartic acid-β-hydroxamate, glycyl- d-aspartic acid and cis-2,3-piperidine dicarboxylic acid. Kinetic parameters for each substrate in 50 mM KPi, pH 7.4, 25°C are reported.

6. ラット唾液腺における D-Amino acid oxidase と D-Aspartate oxidase の細胞内存在様式(第 59 回九州歯科学会総会講演抄録)

6. ラット唾液腺における D-Amino acid oxidase と D-Aspartate oxidase の細胞内存在様式(第 59 回九州歯科学会総会講演抄録)

C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal.

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivo and in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP-PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP-PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

Peroxisomal localization of d-aspartate oxidase and development of peroxisomes in the yeast Cryptococcus humicolus UJ1 grown on d-aspartate

The peroxisomal localization of d-aspartate oxidase (EC. 1.4.3.1) was demonstrated in the yeast Cryptococcus humicolus UJ1 cells grown in the medium containing d-aspartate as a nitrogen source. The conclusion is based on the identical behavior of the enzyme with those of peroxisomal marker enzymes, catalase and urate oxidase, during all steps of subcellular fractionations. Supporting evidence was provided by the morphometric analysis of the peroxisomes with electron microscopy, showing that the cells grown on d-aspartate contained more and larger peroxisomes than those grown on l-aspartate, consistent with the 500-fold and 3-fold higher contents of d-aspartate oxidase and catalase activities, respectively, in the former cells than the latter.

D-Aspartate Oxidase and Free Acidic d-Amino Acids in Fish Tissues

We describe the occurrence of d-aspartate oxidase activity and free d-aspartate and d-glutamate in several tissues of five fish species, carp (Cyprinus carpio), crucian carp (Carassius auratus langsdorfii), rainbow trout (Oncorhynchus mykiss), yellowtail (Seriola quinqueradiata) and sea bream (Pagrus major). N-methyl-d-aspartate was the best enzyme substrate, followed by d-aspartate and d-glutamate. The highest specific activity was observed in the liver, followed by the kidney and brain in the crucian carp and rainbow trout. In the other fishes, enzyme activities were similar in the liver and kidney and lower in the brain except the yellowtail. d-Aspartate (% of total) and d-glutamate (%) were detected with HPLC analysis in the tissues. With the exception of spleen of some species, d-glutamate concentration tended to be higher than those for d-aspartate, but d/(d+ l) ratios were higher for aspartate.

Structural and functional characterization of the human brain D-aspartate oxidase.

D-Aspartate oxidase (DDO) cDNAs were isolated from the human brain RNA using the RT-PCR method. Two forms (DDO-1 and DDO-2) of DDO mRNA were detected. Structural analysis of the DDO cDNAs and genomic DNA showed that DDO-1 and DDO-2 are produced by alternative splicing from a single gene. A protein encoded by the DDO-1 cDNA consists of 341 amino acids, and the amino acid sequence of DDO-2 was identical to that of DDO-1 except for the absence of 59 amino acids covering residues 95-153 of DDO-1. A homogenous preparation of DDO-1 was obtained using an expression system in Escherichia coli. DDO-1 selectively catalyzed the oxidative deamination of D-aspartate and its N-methylated derivative, N-methyl D-aspartate; the values of K(m) and k(cat) for D-aspartate were 2.7 mM and 52.5 mol D-aspartate oxidized x s(-1) x mol(-1) and those for N-methyl D-aspartate were 6.8 mM and 37.7 mol N-methyl D-aspartate oxidized x s(-1) x mol(-1), respectively.

CDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex.

The isolation and sequencing of the complete cDNA coding for a d-aspartate oxidase, as well as the overexpression of the recombinant active enzyme, are reported for the first time. This 2022 bp cDNA, beside the coding portion, comprises a 5' untranslated tract and the whole 3' region including the polyadenylation signal and the poly(A) tail. The encoded protein comprises 341 amino acids, with the last three residues (-Ser-Lys-Leu) representing a peroxisomal targeting signal 1 (PTS1), hitherto unknown for this protein. The overexpression of recombinant d-aspartate oxidase was achieved in a prokaryotic system, and a soluble and active enzyme was obtained which accounted for about 10% of total bacterial protein. Comparisons with the known cDNAs for mammalian d-amino acid oxidase, another peroxisomal enzyme, are also made. The close structural and functional similarities shared by these enzymes at the protein level are not reflected at the nucleic acid level.

D-aspartate localizations imply neuronal and neuroendocrine roles.

Though L-amino acids predominate in living organisms, substantial levels of free D-serine and D-aspartate occur in mammals, especially in nervous and endocrine tissues. Using an antibody specific for glutaraldehyde-fixed D-aspartate, we have localized D-aspartate in rat tissues. In the brain we observe discrete neuronal localizations of D-aspartate, especially in the external plexiform layer of the olfactory bulb, hypothalamic supraoptic and paraventricular nuclei, the medial habenula, and certain brainstem nuclei. In rats 3-4 weeks old, we observe D-aspartate in septal nuclei and in a subset of stellate and basket cells of the cerebellum. D-aspartate is also concentrated in glands, including the epinephrine cells of the adrenal medulla, the posterior pituitary, and the pineal gland. Levels in the pineal gland are the highest of any mammalian tissue. D-aspartate oxidase, visualized by enzyme histochemistry, is concentrated in neurons of the hippocampus, cerebral cortex, and olfactory epithelium, as well as choroid plexus and ependyma. Localizations of D-aspartate oxidase are reciprocal to D-aspartate, suggesting that the enzyme depletes endogenous stores of the amino acid and might inactivate synaptically released D-aspartate.

Distribution of d-aspartate oxidase and free d-glutamate and d-aspartate in chicken and pigeon tissues

1. The presence of d-aspartate oxidase activity and d-glutamate was demonstrated for the first time in several tissues of chicken and pigeon. 2. The identification of the enzyme was based on substrate and inhibitor specificity of the activity as well as other requirements for the activity, and the amino acid was identified with HPLC analysis. 3. In each animal, the highest activity was found in the kidney. In chicken, the hepatic, renal and pancreatic activities were significantly higher in male than female. In pigeon, however, any significant gender difference was not observed. 4. A substantial amount of d-glutamate, as well as d-aspartate, was detected in each tissue, irrespective of species. 5. The contents of the free D-enantiomers were significantly different between two genders in the chicken kidney, heart and pancreas and pigeon liver and kidney. However, any common relationship was not observed between the contents and the D-aspartate oxidase activity.

Light and electron microscopic localization of D-aspartate oxidase in peroxisomes of bovine kidney and liver: an immunocytochemical study.

D-Aspartate oxidase (EC 1.4.3.1; D-ASPOX) specifically oxidizes the D-isomers of dicarboxylic amino acids such as aspartic or glutamic acid. Subcellular fractionation experiments in the past showed its association with peroxisome preparations in kidney cortex and liver. However, no information exists on the in situ localization and distribution of the enzyme in different cell types. We have purified the enzyme from the bovine kidney and raised an antibody against it in rabbits. The monospecificity of the antibody has been confirmed by Western blotting and it does not crossreact with D-amino, acid oxidase. Immunohistochemical localization of the antigen in bovine kidney and liver with the streptavidin-biotin-peroxidase technique revealed a punctate localization in the epithelial cells of proximal nephron tubules, particularly in the straight P-3 segment, as well as in hepatocytes. This is consistent with a localization in peroxisomes. Best results have been obtained with Carnoy-fixed material after paraffin embedding or after fixation with formaldehyde-glutaraldehyde in cryostat sections. Immunoelectron microscopy with protein A-gold confirms the peroxisomal localization of D-ASPOX. Gold particles are distributed over the matrix, suggestive of a peroxisomal matrix enzyme. This is the first report on the localization of D-ASPOX, a little-known peroxisomal enzyme. The techniques described and the antibody prepared will now allow systematic investigation of its tissue distribution.

Purification and properties of d-aspartate oxidase from Cryptococcus humicolus UJ1

d-Aspartate oxidase (EC 1.4.3.1), which is highly specific to d-aspartate, was inducibly produced by a yeast strain which was isolated from soil and identified as Cryptococcus humicolus UJ1. The enzyme was purified to homogeneity as indicated on SDS-polyacrylamide gel electrophoresis. The molecular mass of the monomer subunit was determined to be 40 kDa. The native enzyme was suggested to be a homotetramer by its behavior on gel filtration. The enzyme was shown to be a flavoprotein by its absorption spectral properties, and the flavin was found to be tightly, but not covalently, bound FAD. The purified preparation had a specific activity of 76.1 μmol/min per mg protein with d-aspartate as substrate. Optimum pH was 7.5 and optimum temperature was around 35°C. d-Glutamate was a very poor substrate for the enzyme. N- Methyl- d-aspartate was better than d-glutamate as substrate but markedly poorer than d-aspartate. Malonate was the most effective competitive inhibitor of the compounds tested. The N-terminal amino-acid sequence of the enzyme showed a significant homology with those of d-aspartate oxidases from beef kidney and Octopus vulgaris and those of d-amino-acid oxidases from various sources.