D-amino Acid Oxidase Activity Research Articles

Developmental patterns of peroxisomal enzymes in amphibian liver during spontaneous and triiodothyronine-induced metamorphosis

1. 1. Liver catalase, d-amino acid oxidase, urate oxidase of Alytes obstetricans and Xenopus laevis (anuran amphibians) and fatty acyl-CoA oxidase of Alytes were present at all post-embryonic stages. 2. 2. Catalase and d-amino acid oxidase activities increased during spontaneous metamorphosis of the two species. 3. 3. During triiodothyronine-induced metamorphosis of Alytes larvae, catalase and d-amino acid oxidase activities increased after a latent period. 4. 4. Our results suggest that expression of some hepatic peroxisomal enzymes is modulated by thyroid hormones.

D-Amino acid oxidase in mouse liver—II

1. 1. Activity of d-amino acid oxidase was detected in tissue extract of mouse liver by two sensitive spectrophotometric methods. 2. 2. The activity was also detectable in extracts of the heart, but not of lung.

Two spectrophotometric assays for D-amino acid oxidase: for the study of distribution patterns.

1. Two simple, rapid and sensitive methods are described for measurement of D-amino acid oxidase (DAO) activity using crude tissue extracts for the study of distribution patterns. 2. They detect products of oxidative deamination of D-amino acids catalyzed by DAO, i.e. alpha-keto acids and H2O2 by measuring light absorbances at 445 and 500 nm, respectively. 3. Reliability of the methods was confirmed by quantitative detection of DAO added to the tissue extract, based on standard curves, time courses and values of Km and Ki, obtained using D-Ala as substrate.

Comparative utilization of d- and l-methionine by the white pekin duckling ( Anas platyrhynchos)

1. 1. White Pekin ducklings ( Anas platyrhynchos) were fed a sulfur amino acid-deficient corn-peanut meal basal diet supplemented with either d- or l-methionine (MET) at levels of 0,0.04, 0.08, or 0.16%. 2. 2. Although liver and kidney d-amino acid oxidase activities were induced in birds fed d-MET, their weight gains were only approximately 78% of those fed l-MET at each level of supplementation. 3. 3. Ducklings fed the basal diet supplemented with 0.08 or 0.16% d-MET had significantly ( P<0.05) higher plasma MET levels than birds fed an equimolar amount of l-MET. 4. 4. It was concluded that d-MET was a less efficacious source of dietary MET activity than l-MET for White Pekin ducklings.

Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO2 was not effective in enhancing the operational life of the enzyme preparation.

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney.

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.

Intraparticulate localization of peroxisomal carnitine acyltransferases in chick embryo liver.

Hepatic peroxisomes from 20-d-old chick embryo were purified by sucrose density gradient centrifugation. The specific activities of catalase and D-amino acid oxidase (DAAO) in the peroxisomal fraction were 24- and 32-fold, respectively, higher than those in the homogenate. The isolated peroxisomes included activities of 85.5 and 416 nmol/min/mg protein of carnitine palmitoyltransferase (CPT) and carnitine acetyltransferase (CAT), respectively. CPT was easily solubilized by treatment with Tween 20 or sonication from the peroxisomes, being similar in this respect to catalase. On the other hand, CAT was more resistant to solubilization, behaving similarly to DAAO. These results indicate that CPT as well as catalase is located in the peroxisomal matrix in a soluble form, and that CAT as well as DAAO is weakly associated with some insoluble components.

Effects of D-Amino Acids on Growth Rate and Kidney D-Amino Acid Oxidase in Chicks

Studies were conducted on the effects of feeding D-amino acids on growth rate and D-amino acid oxidase (DAAO) in chick kidney. The crystalline amino acid (AA) diet provided seven amino acids either in the L-form or the DL-form at two concentrations (DL- or .5 DL-AA diets) with all diets containing equal amounts of L-amino acids. Weight gains of chicks fed the DL-AA diet were consistently lower than those fed the L- or .5 DL-AA diet. Kidney DAAO activity was significantly higher in chicks fed either the DL-AA or .5 DL-AA diet as compared with the L-AA diet. Kidney DAAO activity was essentially the same in chicks fed the DL- and .5 DL-AA diets. Increasing the nonspecific nitrogen in the diet had no effect in alleviating growth depression of the DL-AA.

Theoretical approaches to d-amino acid oxidase

Several substrates and roles have been proposed for D-amino acid oxidase (E.C. 1.4.3.3.); however, there is no proof that they possess the required characteristics to account for the ubiquity, large amounts and great activity of the enzyme as found in diverse cells and tissues. Based on the similar stereoposition of identically charged atoms and lateral side chain (R) with respect to the alpha-hydrogen atoms in beta-sheet conformation and in D-amino acids, it is proposed that its substrates may include several membrane-related proteins, partially in beta-sheet conformation, whose alpha-hydrogen atoms would be the real object of D-amino acid oxidase catalysis. A monooxygenase-like enzymatic activity of D-amino acid oxidase with these novel substrates is considered, for which the final products are hypothesized to be protein alpha-carbon hydroxyls resulting from the incorporation of one atom of oxygen into the substrate, the other being reduced to water. Alternatively, it is also proposed that D-amino acid oxidase (and possibly other monooxygenase enzymes) would have a hydroperoxide-synthetase activity. In this case, protein alpha-carbon hydroperoxide and not water, but another reduced molecule, would be the final products. The new enzymatic performances of D-amino acid oxidase and the possible role of its potential final products in redox and other biochemical processes are discussed.

Hypolipidemic effect and enhancement of peroxisomal beta-oxidation in the liver of rats by sodium-(E)-3-(4-(3-pyridylmethyl)phenyl)-2-methyl propenoate (OKY-1581), a potent inhibitor of TxA2 synthetase.

The effects of sodium-(E)-3-(4-(3-pyridylmethyl)phenyl)-2-methyl propenoate (OKY-1581) and (E)-3-(4-(imidazolylmethyl)phenyl)-2-propenoic acid (OKY-046), potent inhibitors to thromboxane A2 synthetase, on peroxisomal beta-oxidation and on lipid levels of liver and serum in the rat were studied. When the animals were administered with OKY-1581 at the dose levels of 100 and 500 mg/kg body weight for 2 weeks, the activity of peroxisomal beta-oxidation increased 2.2- and 6.3-fold respectively. Catalase activity increased 1.3-fold, whereas D-amino acid oxidase (DAAO) and urate oxidase activities did not change. Carnitine acetyltransferase and carnitine palmitoyltransferase activities also increased 2.2- - 4.1-fold and 2.7- - 4.2-fold respectively. These changes of the enzymes related to lipid metabolism were also confirmed by the results of a cell fractionation study. Moreover, the induction of peroxisome proliferation-associated polypeptide having a molecular weight of 80000, which is a bifunctional enzyme in the peroxisomal beta-oxidation system was also observed electrophoretically in the light mitochondrial fraction of the liver of OKY-1581-treated rat. The contents of triglyceride and cholesterol in the serum decreased. These results indicated that the action of OKY-1581 in enhancing hepatic peroxisomal-oxidation is similar to that of a potent hypolipidemic peroxisome proliferator such as clofibrate. On the other hand, differing from OKY-1581, OKY-046 at the dose level of 500 mg/kg for 2 weeks showed no effect on serum and liver lipid levels and on the activities of the peroxisomal enzymes, including a cyanide-insensitive fatty acyl-CoA oxidizing system and carnitine acetyl transferase.

Differentiation of liver peroxisomes in the foetal and newborn rat. Cytochemistry of catalase and D-aminoacid oxidase

Organules containing cytochemically detectable amounts of catalase and D-aminoacid oxidase activities are observed between the 14th and 21st day of development in the parenchymal cells of the foetal rat liver and in the liver of newborn rats. As early as 14 to 15 days, a limited number of small microperoxisomes, scattered in the cytoplasm of very few hepatocytes, can be found. These are roundish shaped, have a granulous matrix and contain very low, hardly detectable levels of the above mentioned enzymes. In later development both the size and the enzymatic content of the organules gradually increase, approaching adult levels at the end of foetal development. Starting from the 18th to 19th day of intrauterine life nucleoids can be seen in many peroxisomes. The morphological and biochemical maturation from microperoxisomes to peroxisomes is accompanied by a gradual increase in the number of stainable organules, both per individual cell and per tissue area.

A sensitive spectrophotometric assay for peroxisomal acyl-CoA oxidase.

A simple spectrophotometric assay was developed for peroxisomal fatty acyl-CoA oxidase activity. The assay, based on the H2O2-dependent oxidation of leuco-dichlorofluorescein catalysed by exogenous peroxidase, is more sensitive than methods previously described. By using mouse liver samples, cofactor requirements were assessed and a linear relationship was demonstrated between dye oxidation and enzyme concentration. By using this assay on subcellular fractions, palmitoyl-CoA oxidase activity was localized for the first time in microperoxisomes of rat intestine. The assay was also adapted to measure D-amino acid oxidase activity, demonstrating the versatility of this method for measuring activity of other H2O2-producing oxidases.

Effects of some anti-inflammatory drugs on biochemical values and on hepatic peroxisomal enzymes of rat.

Effects of tolmetin, diclofenac Na, fenbufen, alclofenac, aminopyrine, mepirizole, thiaramide and aspirin as a positive control, which are widely used in this country as anti-inflammatory drugs, and on body and liver weights, triglyceride and cholesterol level and hepatic peroxisomal enzymes of normolipemic rats were examined. All of these drugs except diclofenac Na affected the enzyme composition of hepatic peroxisomes. Tolmetin (100 mg/kg) and fenbufen (50 mg/kg) increased carnitine acetyltransferase (CAT) and fatty acyl-coenzyme A oxidizing system (FAOS) activities, which participate in hepatic lipid metabolism. The latter also increased the activity of D-amino acid oxidase slightly. Alclofenac (300 mg/kg) increased the activities of FAOS, CAT and carnitine palmitoyltransferase which has been known as the rate-limiting enzyme of fatty acid oxidation in mitochondria, and decreased those of catalase and urate oxidase. Aminopyrine (300 mg/kg) increased the activities of catalase and FAOS. However, none of the above drugs influenced liver weight, serum or liver lipid levels. Mepirizole (300 mg/kg) increased the activities of FAOS and CAT about 2-fold, whereas the activities of catalase and urate oxidase and serum triglyceride level were decreased. Furthermore, these drugs showed no enhancement of the biosynthesis of peroxisome proliferation associated polypeptide having a molecular weight of 80000. From these results, it is concluded that although these drugs have an influence on the enzyme composition of hepatic peroxisomes, they may not induce the peroxisome population in hepatic cells. Thus, the possibility of hepatocarinogenicity and lipid lowering effect through the peroxisome-proliferation would be excluded.

Histochemical staining of cells containing flavoenzyme D-amino acid oxidase based on its enzymatic activity: Application of a coupled peroxidation method.

A histochemical method for the demonstration of D-amino acid oxidase activity in rat kidney, liver and brain is described. The method is a peroxidase-coupled procedure, which is based on the intensifying effect of nickel ions on 3, 3′-diaminobenzidine-based product formation with peroxidase. The substrates used were D-proline and D-alanine, of which the former showed more intense staining. The histochemical reaction was suppressed by benzoate, a competitive inhibitor. There was no staining on omission of a substrate or peroxidase from the incubation medium. The method was specific for D-amino acid oxidase. The oxidase in kidney and brain showed resistance to the fixatives used (e. g. 8% glutaraldehyde), while the enzyme in hepatocytes did not. The reaction product was black, electron dense, stable and strictly confined to cells containing the oxidase. The method was successfully applied to the histochemical identification of the type of cell containing the oxidase at both the levels of light and electron microscopy. The oxidase activity was shown to be present in hepatocytes in the liver, proximal tubule cells in the kidney, and only astrocytes, including Bergmann glial cells, in the brain. In the midbrain, the oxidase was almost exclusively restricted to the tegmentum.

D-amino acid oxidase fromTrigonopsis variabilis: Comparison of enzyme specificity ?in vivo? and ?in vitro?

A method is described for permeabilization of intact cells of the yeastTrigonopsis variabilis with respect toin vivo measuring D-amino acid oxidase activity. The kinetic results so obtained differ from those obtained with the purified enzyme, pointing to the advantage of using the purified enzyme or the permeabilized cells in the oxidative deamination of different D-amino acids.

Studies of D-amino acid oxidase activity in human epidermis and cultured human epidermal cells.

The occurrence of the enzyme D-amino acid oxidase in human epidermis and cultured epidermal cells was investigated. When explant cultures of human epidermis were cultured in a medium containing D-valine instead of L-valine and supplemented with undialyzed serum, good growth of both epithelial cells and fibroblasts was observed. However, when the serum was dialyzed neither cell type could be cultured in D-valine medium indicating the absence of D-amino acid oxidase in both cell types. When epithelial cultures initiated in L-valine medium were changed to D-valine medium after 1-2 weeks, growth stopped immediately, and the epithelial cells showed signs of extensive degeneration, indicating that skin epithelial cells have a very low endogenous pool of L-valine. When these cultures were re-fed L-valine medium after 2 weeks in D-valine medium, this resulted within a few days in the reappearance of epithelial outgrowth. The activity of D-amino acid oxidase in human epidermal cells was further studied by histochemistry and in homogenates of epidermis and cultured epidermal cells. Whereas high activity of histidase was observed in the epidermal cells, no activity of D-amino acid oxidase could be detected. This report shows that D-amino acid oxidase does not serve as a market enzyme for epithelial cells in general as previous tissue culture studies might indicate. Human skin epithelial cells do not contain detectable amounts of D-amino acid oxidase activity, and therefore, D-valine medium is not suitable for selection of growth of skin epithelial cells in culture.

Peroxisomes in the liver of frog, Bombina orientalis.

The existence of peroxisomes was investigated in the liver of frog, Bombina orientalis, which characteristically cannot degrade cholesterol further than to C27 higher bile acids such as 3α, 7α, 12α-trihydroxy-5β-cholestanoic acid (THCA) or 3α, 7α-dihydroxy-5β-cholestanoic acid (DHCA). The activities of catalase, D-amino acid oxidase, urate oxidase, and the CN-insensitive fatty acyl-CoA β-oxidizing system (FAOS) were used as markers of peroxisomes. Catalase activity was present to almost the same extent in Bombina liver as in rat liver. D-Amino acid oxidase and urate oxidase were also present in the frog liver, but the activities were much lower than those in the rat liver. In a cell fractionation experiment, the highest specific activities of catalase, D-amino acid oxidase, urate oxidase and CN-insensitive FAOS were all found in the light mitochondrial fraction, and upon sucrose density gradient centrifugation, these activities were concentrated in the density fraction around 1.21-1.22. On the other hand, the densities of mitochondria and lysosomes were 1.19 and 1.20, respectively. From the above results, we concluded that Bombina liver contains peroxisomes. Next, the characteristics of CN-sensitive (mitochondrial) or CN-insensitive (peroxisomal) FAOS and carnitine acyltransferase activities were compared in the partially purified peroxisomes and mitochondria using various chain length fatty acyl-CoAs (C2-C20). FAOS activities of the peroxisomes were effective for long-chain fatty acyl-CoAs (C8-C16), while the mitochondrial FAOS showed broad specificity for short- to long-chain fatty acyl-CoAs. Both the peroxisomes and the mitochondria showed high carnitine acyltransferase activities for acetyl-CoA and C8 to C12 fatty acyl-CoAs. Administration of clofibrate for 7 d enhanced the activities of catalase and CN-insensitive FAOS, but the D-amino acid oxidase activity decreased and the urate oxidase activity was unchanged. The results indicate that peroxisomes do exist in Bombina liver, but the particles may be involved primarily in the exhaustive degradation of general fatty acids, not in the synthesis of bile acids. When necessary, the mitochondria and peroxisomes may act cooperatively in order to obtain energy from various fatty acids.

Coat color genes of D-amino acid oxidase deficient ddY/DAO- mice and nonlinkage of gene for D-amino acid oxidase to coat color genes.

Coat color genes of mutant ddY/DAO- mice that do not have D-amino acid oxidase were examined by crosses of these mice with NC (AAbbCC) and II TES (aabbCCddss) mice. All the (ddY/DAO-×NC)F1 mice had agouti coats and the (ddY/DAO-×II TES)F1 mice had black coats. These results indicate that the ddY/DAO- mice carry aaBBcc DDSS gene.Linkage relationship of Dao-1 gene coding for D-amino acid oxidase to coat color genes was examined. Segregants produced from backcrosses of (ddY/DAO-×IITES)F1 with ddY/DAO- and (ddY/DAO-×NC)F1 with ddY/DAO- were examined for D-amino acid oxidase activity and coat color. F2 segregants from a (ddY/DAO-×NC) cross were also studied. These crosses showed that Dao-1 was not linked to a, b or c.

Characterization of glyoxysomes in yeasts and their transformation into peroxisomes in response to changes in environmental conditions

During growth of the yeasts Candida utilis and Hansenula polymorpha in mineral media containing ethanol as a carbon source and ammonium sulphate as a nitrogen source, the specific activities of isocitrate lyase and malate synthase were significantly increased when compared to glucose/ammonium sulphate-grown cells. In addition to the enhanced levels of these glyoxylate cycle enzymes, an increase in the specific activities of d-amino acid oxidase, amine oxidase or urate oxidase was observed when ammonium sulphate in the ethanol medium was replaced by d-alanine, methyl- or ethylamine, or uric acid. The subcellular localization of these enzymes was investigated by cell fractionation studies involving homogenization of protoplasts followed by differential and sucrose gradient centrifugation. In ethanol/ammonium sulphate-grown cells, isocitrate lyase and malate synthase cosedimented in a fraction together with catalase and part of the malate dehydrogenase. Electron microscopy revealed that this fraction consisted of microbodies which must be regarded as glyoxysomes. Two other glyoxylate cycle enzymes, citrate synthase and aconitase together with the other part of malate dehydrogenase, cosedimented with cytochrome c oxidase, a mitochondrial marker enzyme. In ethanol/d-alanine-, ethanol/methylamine- or ethanol/ethylamine-grown C. utilis and ethanol/uric acid-grown H. polymorpha, a peroxisomal enzyme, i.e. d-amino acid oxidase, amine oxidase or uric acid oxidase cosedimented with the glyoxysomal key enzymes. Cytochemical staining experiments demonstrated that in these variously-grown cells the activities of the oxidases were confined to the microbodymatrix; this also contained malate synthase activity.

Peroxisomal fatty acyl-coenzyme A oxidation in chicken liver

The activities of antimycin A-insensitive palmitoyl-CoA oxidation and of palmitoyl-CoA oxidase in peroxisomes from chicken liver were similar to those of rat liver. Catalase and d-amino acid oxidase activities in peroxisomes from chicken liver were lower than those of rat liver and urate oxidase was not detected. Carnitine acetyltransferase and palmitoyltransferase levels in chicken liver were 18- and 2-fold higher, respectively, than those of rat liver. Peroxisomal palmitoyl-CoA oxidation of chicken liver was inhibited by cyanide, in contrast to that of rat liver, although it was insensitive to antimycin A. Subcellular distribution of this enzyme was similar to that of rat liver; i.e., it was located only in the peroxisomes. The fatty acyl-CoA oxidase had a higher affinity toward medium- to long-chain fatty acyl-CoAs (C 8 to C 16) than shorter-chain analogs. The fatty acyl-CoA dehydrogenase had a broad affinity toward fatty acyl-CoAs (C 4 to C 18). Carnitine acetyltransferase was distributed equally in both peroxisomes and mitochondria. Carnitine palmitoyltransferase was distributed in the proportion of 20 and 80% in peroxisomes and mitochondria, respectively.