D-amino Acid Oxidase Activity Research Articles

Gas chromatographic characterization of free d-amino acids in the blood serum of patients with renal disorders and of healthy volunteers

A capillary gas chromatographic method, using the chiral stationary phase Chirasil-L-Val, after treatment and isolation with Dowex 50W X8 cation exchanger and conversion into trifluoroacetyl-1-propyl esters or pentafluoropropionyl-1 (or 2)-propyl esters, has been developed for the determination of the relative amounts of free d-amino acids in the blood serum of eighteen patients with renal failure (continuous ambulatory peritoneal dialysis (CAPD), n = 11; hemodialysis, n = 5; nephrotic syndrome, n = 2) and compared with data obtained from healthy volunteers ( n = 5). Significant amounts of d-Ala (0.5–13%) and d-Asx (1.5–7.7%; Asx = Asp + Asn) were found in all serum samples. d-Ser was detected in the serum of all patients with renal disorders and, in addition, d-Pro (0.6–2.5%) was found in the serum of all patients undergoing hemodialysis and with nephrotic syndrome. d-Ser (2.9–3.1%) and d-Pro (0.6–0.9%) were also found in the samples of three volunteers. d-Leu (1.2–1.7%) was present in three patients with CAPD, and d-Glx (0.3–1.3%; Glx = Glu + Gln) was present in eight of eighteen patients with renal malfunction. Linear regression analysis of the relative amounts of d-amino acids and the serum creatinine levels of all donors revealed positive correlation factors for d-Asx ( r = 0.748) and d-Ser ( r = 0.667), but not for d-Pro and d-Ala. Remarkably high amounts of d-Ser (12.1 and 19.8%) were found in two hemodialysates investigated. Participation of intestinal bacteria and nutrition are discussed as possible sources of serum d-amino acids. An increase of some d-amino acids in the serum of patients with renal diseases might be explained, in part, by decreased activity of renal d-amino acid oxidase.

Free d-serine, d-aspartate and d-alanine in central nervous system and serum in mutant mice lacking d-amino acid oxidase

We have examined whether d-amino acid oxidase (DAO) regulates free d-serine content using mutant ddY/DAO − mice lacking DAO activity. We find that the content of d-serine in the serum and cerebellum of mutant mice is much higher than that of normal mice, whereas a slight but significant difference in the cerebral d-serine level is observed between the two strains. These results suggest that, although DAO may participate in the catabolism of d-serine in the cerebellum and periphery, there appears to be other mechanisms for catabolism of endogenous d-serine in the brain.

The use of unfixed cryostat sections for electron microscopic study of D-amino acid oxidase activity in rat liver.

Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium containing D-proline as substrate and cerium ions as capture reagent for hydrogen peroxide. After an incubation period of 30 min, ultrastructural morphology was retained to such an extent that the final reaction product could be localized in peroxisomes, whereas the crystalline core remained unstained. Control incubations were performed in the absence of substrate; the lack of final reaction product in peroxisomes indicated the specificity of the reaction. We conclude that the semipermeable membrane technique opens new perspectives for localization of enzyme activities at the ultrastructural level without prior tissue fixation, thus enabling localization of the activity of soluble and/or labile enzymes.

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.

A single-base-pair substitution abolishes d-amino-acid oxidase activity in the mouse

Mutant ddY/DAO − mice lacking d-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO − mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was clone into a plasid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G→A) which causes a Gly-181→Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. THe transfeceted cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicated that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO − mice.

Water-soluble vitamin requirements of tilapia. 2. Riboflavin requirement of blue tilapia, Oreochromis aureus

Juvenile tilapia, Oreochromis aureus, of initial average weight 0.71 g were fed six diets supplemented with different levels of riboflavin (0, 3, 6, 12, 24, and 48 mg/kg) for 10 weeks in flow-through aquaria at a water temperature of 27.0 ± 1 °C. Fish fed the riboflavin-free diet performed poorly in terms of growth parameters and exhibited typical signs of riboflavin deficiency such as lethargy, fin erosion, anorexia, loss of normal body color, short body dwarfism and a high incidence of cataracts. Fish fed the riboflavin-free diet were also anaemic. No deficiency signs were observed in fish fed the riboflavin-supplemented diets. No significant difference was observed in weight gain of the fish fed the riboflavin-supplemented diets; however, feed efficiency was improved at the 6 mg riboflavin/kg diet level. Hepatic D-amino acid oxidase activity was low in the riboflavin-deficient fish and increased in a dose-response manner with maximum activity being observed in fish fed the 6 mg riboflavin/kg diet. Based on these data, the dietary riboflavin requirement for blue tilapia was determined to be 6 mg riboflavin/kg of diet.

Granule-associated flavin adenine dinucleotide (FAD) is responsible for eosinophil autofluorescence.

Unstained human eosinophils exhibit marked autofluorescence in comparison to other leukocytes due to a granule-associated fluorescent substance. Fluorescence spectroscopy of granule extracts reveals excitation maxima at approximately 380 and approximately 450 nm with a single emission at approximately 520, characteristic of flavins. The fluorescent material from eosinophil granule extracts was characterized by fluorescence, high-performance liquid chromatographic, and enzymatic analyses. First, acidification to pH 2.6 resulted in increased fluorescence, indicative of flavin adenine dinucleotide (FAD). Second, because flavin mononucleotide (FMN) and riboflavin cannot be distinguished by acidification, high-performance liquid chromatography was performed and revealed a predominance of FAD and smaller amounts (< 15%) of both FMN and riboflavin. Third, the presence of FAD was clearly demonstrated by reconstitution of the activity of D-amino acid oxidase, a FAD-dependent enzyme, when granule extracts were added to the apoenzyme. Thus, we have identified FAD as the predominant fluorophore in eosinophil granules. The small amounts of FMN and riboflavin detected may result from the hydrolysis of FAD under the acidic conditions of granule extraction. Because fluorescent material is deposited onto target cells by eosinophils, it is possible that granule-associated flavoproteins may act as a source of hydrogen peroxide and/or superoxide, which, in conjunction with eosinophil peroxidase, could yield potent cytotoxic agents.

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.

Enzymic and chemical conversions of cephalosporin C to 7-(glutarylamido)cephalosporanic acid

The resting cells of Trigonopsis variabilis having high d-amino acid oxidase activity were used to convert cephalosporin C into 7-(α-ketoadipylamido)cephaeosporanic acid. Treatment of the latter compound with the same equivalent of hydrogen peroxide quantitatively yields a compound whose physicochemical properties are similar to those of 7-(glutarylamido)cephalosporanic acid synthesized from glutaric anhydride and 7-aminocephalosporanic acid. The overall yield of 7-(glutarylamido)cephalosporanic acid from enzymic and chemical reactions was 52% of the theoretical amount.

High-performance liquid chromatographic determination of d-amino acid oxidase activity

A new procedure for the assay of D-amino acid oxidase activity has been developed. alpha-Ketoisovaleric acid, derived from D-valine, was estimated by high-performance liquid chromatography after reaction with o-phenylenediamine to give the corresponding quinoxalinol derivative. alpha-Ketovaleric acid was used as an internal standard to ensure the reproducibility of the method. As an example of application, kidney cortex homogenates were analyzed for their D-amino acid oxidase activity. The advantages of the presented procedure for the determination of the enzymatic activity in biological samples compared with previously reported procedures are discussed.

Peroxisomal oxidases and catalase in liver and kidney homogenates of normal and di(ethylhexyl)phthalate-fed rats

1. 1. Activities of peroxisomal oxidases and catalase were assayed at neutral and alkaline pH in liver and kidney homogenates from male rats fed a diet with or without 2% di(2-ethylhexyl)phthalate (DEHP) for 12 days. 2. 2. All enzyme activities were higher at alkaline than at neutral pH in both groups. 3. 3. The effect of the DEHP-diet on the peroxisomal enzymes was different in kidney and liver. Acyl-CoA oxidase activity was raised three-and sixfold in kidney and liver homogenates, respectively. The activity of d-amino acid oxidase decreased in liver, but increased in kidney homogenates. In liver homogenates, urate oxidase activity was not affected by the DEHP diet. The catalase activity was twofold induced in liver, but not in kidney. 4. 4. The differences suggest that the changes of peroxisomal enzyme activities by DEHP treatment are not directly related to peroxisome proliferation. 5. 5. DEHP treatment caused a marked increase of total and peroxisomal fatty acid oxidation in rat liver homogenates. 6. 6. In the control group the rate of peroxisomal fatty acid oxidation was higher at alkaline pH than at neutral pH. 7. 7. This rate was equal at both pH values in the DEHP-fed group, in contrast to the acyl-CoA oxidase activity. These results indicate that after DEHP treatment other parameters than acyl-CoA oxidase activity become limiting for peroxisomal β-oxidation.

Effect of Respiratory Infection on Tissue Riboflavin and Flavin Enzymes in Mice

The effects of Klebsiella pneumoniae infection on red blood cell and liver riboflavin levels and a few flavin enzymes were examined to verify our earlier hypothesis based on human experience that upper respiratory infections result in mobilization of riboflavin from tissues into blood, resulting in a rise in red blood cell riboflavin and an increased saturation of erythrocyte glutathione reductase (EGR) with its coenzyme flavin adenine dinucleotide (FAD; reduction in EGR activation coefficient, AC, values). Thirty-six-day-old male mice fed a diet marginally sufficient in riboflavin were injected with a single sublethal dose of K. pneumoniae. Batches of control, deficient infected and deficient uninfected animals were killed during the peak period of infection and after recovery. Infection brought about a significant rise in basal EGR activity due to greater saturation with coenzyme FAD and a concomitant reduction in EGR-AC values. On recovery, basal EGR activity decreased, and EGR-AC values increased to levels beyond control values, suggesting riboflavin deficiency. In the liver, acyl-coenzyme A dehydrogenase showed a significant decrease and pyridoxamine-phosphate oxidase activity a significant increase. Both the enzymes normalized after recovery. D-amino acid oxidase activity did not change.

Differential effect of sunflower seed oil on hepatic and renal d-amino acid oxidase in the rat

1. 1. The specific activity of hepatic and renal peroxisomal d-amino acid oxidase ( d-Aaox) was measured in rats fed diets containing various quantities of vegetable oil. 2. 2. Increasing the amount of dietary sunflower seed oil (SSO) from 10 to 25% (w/w) reduced the specific activity of hepatic d-Aaox by up to 30% after 10 days. 3. 3. In both tissues, the enzyme activity was moderately decreased during the first two-day period after administration of the 25% SSO diet was begun. Unlike hepatic d-Aaox, renal d-Aaox returned to its baseline level in the kidney after the third day. 4. 4. In contrast to SSO, hydrogenated coconut oil (HCO) did not evoke alterations of d-AAOX activity. 5. 5. The activity levels of another peroxisomal enzyme, l-2-hydroxy acid oxidase ( l-Haox), in the liver of rats fed the high-SSO diet vs those fed the control diet were similar. 6. 6. The subcellular distribution of d-Aaox and d-Haox was not altered in the liver of rats fed the 25% SSO diet during the 10-day period.

Peroxisomes in the head of Drosophila melanogaster

Peroxisomes were localized in the head of wild-type and mutant strains of Drosophila melanogaster by use of a cytochemical method for the demonstration of d-amino acid oxidase activity. With similar techniques we had found previously that vertebrate photoreceptors have few, if any, bodies with cytochemically demonstrable oxidase activity, but that the pigment epithelial cells adjacent to the photoreceptors have a substantial population of such bodies. Peroxisomes in Drosophila were very abundant in the fat body. Probable peroxisomes were also present in the peripheral retina of the eye, including in retinular (retinula) and pigment cells, but there were very few of them. Thus, our results suggest that the fat body, which lies adjacent to the eye, is the principal site of peroxisomal function in the head. Peroxisome functions in the Drosophila head may include participation in the genesis of eye pigments.

D-aspartate oxidation by rat and bovine renal peroxisomes: an electron microscopic cytochemical study.

D-amino acid oxidase, a peroxisomal enzyme, and D-aspartate oxidase, a potential peroxisomal enzyme, share biochemical attributes. Both produce hydrogen peroxide in flavin-requiring oxidative reactions. Such similarities suggest that D-aspartate oxidase may also be localized to peroxisomes. Definitive identification of D-aspartate oxidase as a peroxisomal enzyme depends, however, on visualization at the electron microscopic level. Using incubation conditions shown to be specific for the enzyme in biochemical studies, this report extends the cytochemical localization of D-amino acid oxidase to bovine renal peroxisomes, and shows that D-aspartate can be oxidized by rat and bovine renal peroxisomes. An unexpected finding was the sensitivity of both D-amino acid oxidase activity (proline specific) and D-aspartate oxidase activity to inhibition by agents used in biochemical studies to discriminate between the two enzyme activities. Therefore, it is possible that, in the cytochemical system used in this study, (a) either D-proline and D-aspartate are substrates for only one enzyme or (b) the two enzymes have additional overlapping biochemical properties.

Histochemistry of reactive oxygen-species (ROS)-generating oxidases in cutaneous and mucous epithelia of laboratory rodents with special reference to xanthine oxidase

Cutaneous and mucous epithelia of various organs of laboratory rodents were analysed histochemically for reactive oxygen species (ROS)-generating oxidases using cerium methods. High activities of xanthine oxidase and also superoxide dismutase were present in orthokeratotic stratified squamous epithelia of skin, lips, esophagus and forestomach and parakeratotic keratinizing stratified epithelia of vagina, tongue and penis. Moreover, activity was found in simple epithelium of the uterus and intestine of rats, mice and guinea-pigs. Moderate activities of monoamine oxidase and D-amino acid oxidase were only seen in enterocytes of large and small intestine, whereas alpha-hydroxy acid oxidase could not be detected at all. With the use of specific inhibitors for superoxide anions-producing xanthine oxidase and H2O2-generating superoxide dismutase it was shown that epithelial cells of all studied external and internal surface epithelia contain a highly effective xanthine oxidase-superoxide dismutase system. It is hypothesized that this system might have a general microbicidal function and might play a special role in tumor promotion of the skin.

Riboflavin requirements in on-growing rainbow trout, Oncorhynchus mykiss

Rainbow trout ( O. mykiss) of 60 g mean weight were given, for 18 weeks, diets containing 0.6, 1.6, 2.7, 5.6 and 12.9 mg riboflavin/kg dry diet. A level of 2.7 mg riboflavin/kg dry diet was required to: (a) prevent the appearance of gross deficiency symptoms including ocular opacities; (b) obtain maximal riboflavin levels in liver and heart; (c) maximal activity of hepatic D-amino acid oxidase (D-AAO); (d) minimal stimulation of the activities of D-AAO, erythrocyte glutathione reductase (EGR) and hepatic glutathione reductase (HGR), after “in vitro” pre-incubation with the coenzyme flavin adenine dinucleotide (FAD). Hepatic D-AAO activity was shown to be a reliable indicator of incipient riboflavin deficiency and EGR a convenient indicator of riboflavin deficiency in the field.

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.

Detection of 2H-1,4-thiazine-5,6-dihydro-3-car☐ylic acid (aminoethylcysteine ketimine) in the bovine brain

2H-1,4-Thiazine-5,6-dihydro-3-car☐ylic acid (trivial name : aminoethylcysteine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts by means of three different procedures. Gas liquid chromatography of protein-free extracts of five bovine brains revealed the presence of this compound at concentrations ranging from 2 to 3 nmol/g wet weight of tissue. The enzymatic method based on the inhibition of D-amino acid oxidase activity by aminoethylcysteine ketimine together with an high-performance liquid chromatography procedure confirm the identification and quantitations obtained with gas liquid chromatography. The discovery of this compound structurally similar to pipecolic acid opens the question of its physiological role in the central nervous system.

Occurrence of d-alanine in the eggs and the developing embryo of the sea urchin Paracentrotus lividus

1. 1. d-Alanine has been found in appreciable amounts in the eggs and embryos of the sea urchin Paracentrotus lividus. 2. 2. The content of d-alanine, expressed as pmol/egg or embryo, is 1.32 in the egg, 0.81 in the blastula, 0.54 in the gastrula and 0.60 in the pluteus. 3. 3. The percentage of d-alanine with respect to the total alanine ( d + l) decreases during embryonic development. 4. 4. d-Amino acid oxidase, d-alanine transaminase and d-alanine racemase activities were found neither in eggs nor in embryos. 5. 5. Therefore, it does not appear likely that d-alanine is subject to oxidative metabolism. 6. 6. The decrease in this d-amino acid during development may be due to its utilization in the synthesis of a more complex molecule.