D-amino Acid Oxidase Activity Research Articles

Sensitivity of hepatic D-amino acid oxidase and glutathione reductase to the riboflavin status of the rainbow trout ( Salmo gairdneri)

Two hepatic enzymes, D-amino acid oxidase (DAAO) and glutathione reductase (GR), were assessed for their potential to determine the riboflavin requirement of rainbow trout ( Salmo gairdneri). Both enzymes exhibit sufficient activity to permit precise quantification in fry, fingerlings and young juveniles. Hepatic GR activity was not affected, even in severely riboflavin-deficient fish. DAAO activity was depressed by riboflavin deficiency, and appeared to provide a more sensitive indicator of incipient vitamin insufficiency than either tissue flavin storage or grossly observable symptoms. DAAO activity was not affected by reduced feed intake as indicated by examination of pair-fed controls. The results suggest that liver maximal DAAO activity may prove useful for determining the riboflavin requirement of young rainbow trout.

Effects of actinomycin D and cycloheximide on the activities of catalase and D-amino acid oxidase in the rat kidney cortex during sodium restriction.

This study was carried out to determine whether actinomycin D and cycloheximide affect the activities of catalase and D-amino acid oxidase in the rat kidney cortex under conditions of sodium restriction. Animals were maintained on a sodium-deficient diet and injected intra-peritoneally with actinomycin D or cycloheximide. Low sodium intake for 7d resulted in increased activities of catalase and D-amino acid oxidase in the kidney cortex. Daily adminis-tration of actinomycin D (20μg·kg-1·d) or cycloheximide (80μg·kg-1·d) with the intake of sodium-deficient diet for 7d had no influence on the amount of protein in the kidney cortex, but preferentially prevented the elevation of the activities of catalase and D-amino acid oxidase by sodium restriction. These results provide evidence for the induction of peroxisomal enzymes by low sodium intake.

Effects of phytol, a branched, long-chain aliphatic alcohol, on biochemical values and on hepatic peroxisomal enzymes of rats.

The effects of phytol on biochemical values (triglyceride and cholesterol levels of liver and serum, body weight gain and liver weight), and peroxisomal enzyme activities were investigated in rats. The rats were given phytol orally at the dose of 200 or 500 mg/kg body weight daily for 3 weeks. The triglyceride and cholesterol levels of the serum of the rats treated with phytol at 500mg/kg decreased by 32 and 35% respectively, while no influence on body weight gain or liver weight was observed. Fatty acyl-CoA oxidase activity and catalase activity in the livers of treated rats were increased by 29 and 20% at 200mg/kg, and by 36 and 44% at 500mg/kg, respectively. D-Amino acid oxidase activity was also increased by 54% (at 200mg/kg) and 85% (at 500mg/kg) in treated animals, but the activity of another peroxisomal enzyme, urate oxidase, did not change. On sucrose density gradient centrifugation of the light mitochondrial fractions prepared from the livers of control and treated rats, similar distribution patterns were observed in both samples, but the activities of catalase and fatty acyl-CoA oxidase of peroxisomal fraction of the treated (500mg/kg) group were increased by 68 and 45%, respectively. These results suggest that phytol might enhance peroxisomal β-oxidation and also induce some peroxisomal enzymes, causing change of peroxisomal enzyme compositions.

Interaction between flavins and alcohols

The effect of alcohols on the spectral properties of riboflavin derivatives in non-polar solvent was studied by various spectroscopic methods in order to support the view point that alcohol may directly interact with the isoalloxazine moiety of FAD and enhance the catalytic activity of D-amino acid oxidase (DAAO). The most likely association complex between alcohol and riboflavin is 1 : 1 stoichiometric complex through the 3-N imino and the 2-C carbonyl groups of the isoalloxazine ring and the hydroxyl group of alcohols. It appears that methanol has a larger association constant than any other alcohols, and the association constant decreases with the increase in carbon number and with the steric requirement of the alkyl group of alcohols.

Membrane-bound flavin adenine dinucleotide in methanobacterium bryantii

Noncovalently attached flavin was isolated and partially purified from the membrane fraction of Methanobacterium Bryantii . The flavin was identified as FAD by absorption and fluorescence spectroscopy, effects on the spectra of reduction and of protonation, phenol extractability, behavior in thin layer chromatography in two solvent systems, and ability to reconstitute activity of the FAD-specific enzyme D-amino acid oxidase. These singular organisms thus are capable of synthesizing isoalloxazine-type flavins as well as the unique 5-deazaflavin factor F 420 . Membrane-bound FAD may thus (in addition to iron-sulfur centers and a nickel species) be involved in energy-coupled methanogenesis.

Effects of some hypolipidemic drugs on biochemical values and on hepatic peroxisomal enzymes of normolipemic rat.

Effects of vitamin B2-butyrate, nicomol, ML-236B, KF 1492 and pantethine, which are hypolipidemic drugs, on biochemical values and on hepatic peroxisomal enzymes of normolipemic rat. 1) Vitamin B2-butyrate (100 mg/kg) and nicomol (lg/Kg) increased carnitine acetyltransferase and D-amino acid oxidase activities, respectively, while these drugs had no influence on body weight, liver weight, serum and liver triglyceride, and serum and liver cholesterol levels. 2) ML-236B (300 mg/kg) had no influence on biochemical values and on activities of peroxisomal enzymes containing catalase. 3) KF 1492 (300 mg/kg) had no influence on the biochemical values, but an increase in the activities of fatty acyl-CoA oxidizing system (FAOS) and carnitine acetyltransferase (CAT) participating hepatic lipid metabolism was observed. 4) Pantethine (lg/kg) had no influence on the biochemical values, except a little decrease in the growth rate. However, increase by about 10% in the activities of urate oxidase and D-amino acid oxidase was observed. Catalase activity was decreased to 60% of control level. From these results, it is concluded that, in contrast to clofibrate, vitamin B2-butyrate, nicomol, ML-236B, KF 1492 and pantethine have little influence on the lipid metabolism of normolipemic animal and on the hepatic peroxisomal enzymes, indicating that the action mechanism of these drugs may be different from that of clofibrate and that the participation of hepatic peroxisomes in hypolipidemic activities of these drugs may be little if any.

Studies on peroxisomes. IX. Biochemical effects of simfibrate on precursor incorporation into polypeptide associated with peroxisome proliferation in rat liver.

Treatment with 1,3-propanediol-bis(2-p-chlorophenoxy-isobutyrate) (simfibrate), a hypolipidemic drug, showed little effect on liver weight and hepatic lipid level. When the effects of simfibrate on rat liver peroxisomal enzymes were examined, it was found that cyanide-insensitive fatty acyl-CoA oxidizing system and carnitine acetyltransferase activity increased from 0.38 U/g liver to 4.5 U/g liver and from 227 U/g liver to 6,450 U/g liver, respectively. The activity of D-amino acid oxidase decreased from 1.05 U/g liver to 0.39 U/g liver. Other peroxisomal enzymes including catalase and urate oxidase were not significantly changed by this drug. Of the protein components of the light mitochondrial fraction of simfibrate-treated rat liver, a polypeptide with a molecular weight of approximately 76,000, which has been suggested to be one of the peroxisomal proteins, increased in the treated rat liver (as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis) and DL-[4,5-3H]leucine-incorporation into this particular component was also stimulated to a level about 3 times that of the control.

Identification and subcellular localization of catalase activity in bovine adrenal medulla and cortex

Catalase activity was detected in homogenates of bovine adrenal cortex and medulla. Analysis by equilibrium density centrifugation in isoosmotic metrizamide-sucrose gradients revealed that 70% of the medullary catalase activity was soluble while most of the remainder was found in a particulate form with a density of 1.175 g/ml. This was distinct from the densities of lysosomes, mitochondria, and chromaffin granules. Catalase activity in adrenal cortex was primarily (90%) soluble with only 6% being particulate, with a density of 1.185 g/ml. d-Amino acid, uric acid, and α-hydroxyacid oxidase activities, often associated with peroxisomes in other tissues, were absent from homogenates and catalase-containing gradient fractions from either cortex or medulla. There was an indication that some catalase activity was associated with chromaffin granules on the basis of density gradient analysis of both medullary homogenates and crude granule preparations. When granule fractions were subjected to osmotic shock, catalase activity distributed between soluble and sedimentable fractions differently from epinephrine and dopamine β-hydroxylase activity. The sedimentable catalase activity remained associated with chromaffin granule membranes upon isopycnic centrifugation. We concluded that catalase activity in both adrenal cortex and medulla was largely cytoplasmic, but that both tissues contained at least some catalase in dense organelles. Catalase activity which may be associated with chromaffin granules represents a small fraction of the total activity in the medulla.

Interactions of progesterone with D-amino acid oxidase—different effects on apo- and holo-enzyme

A simple method using charcoal treatment was developed for the preparation of apo-D-amino acid oxidase from rat kidney homogenates. This apo-D-amino acid oxidase was used to study the effect of progesterone on the apo- and holo-enzyme. Progesterone inhibited the activity of D-amino acid oxidase, when the apo-enzyme, preincubated with saturating amounts of FAD was used; this effect varied with FAD concentration. Progesterone did not inhibit the activity when added to a mixture of non-preincubated apo-enzyme and FAD; this suggests that progesterone has different effects on apo- and holo D-amino acid oxidase.

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.

Effect of triton WR-1339 on peroxisomal enzymes of rat liver

The effect of Triton WR-1339 on peroxisomal enzymes of rat liver was studied. The dose vs. response relationships of peroxisomal enzyme activities to Triton WR-1339 were first examined 3.5 days after injection. Catalase activity was reduced to 50% of that of the control at a dose of 200 mg per 100 g body weight; it was found that the decrease depended on the dose of this compound. Urate oxidase activity was not significantly affected. D-Amino acid oxidase activity showed intermediate behavior. The activities of these enzymes were found to be reduced more markedly at 2 days than at 3.5 days after injection, and subsequently the levels of the activities recovered. At 2 days after injection of a dose of 200 mg per 100 g body weight, the activities of catalase, D-amino acid oxidase and urate oxidase had decreased to 40, 60 and 60%,respectively, of the control values. It was found that the decreases in the activities of these enzymes caused by Triton WR-1339 had occurred in the large granule fraction, but not in the cytoplasm. Measurement of the specific activity, Ouchterlony gel diffusion and quantitative immunoprecipitation suggested that there was a similarity between the Triton WR-1339-treated and untreated rats in the nature of purified catalases. These results suggest that Triton WR-1339 depresses the activities of liver peroxisomal enzymes, especially the catalase activity.

D-Amino acids of the amino acid pool and occurrence of racemase and D-amino acid oxidase activities in Escherichia coli B.

Less than 20% of the amino acid content of the amino acid pool of Escherichia coli B exists in the D-form. Alanine, glutamic acid, and valine were shown by gas- chromatography to be partially in the D-form. Only D-alanine was formed by racemization in the crude extract of this organism. Alanine racemase was easily released from the membranes or vesicles but D-alanine oxidase activity remained firmly bound to the membrane. Most protein amino acids stimulated proline uptake into the vesicles, and the oxidative deamination activities were verified by the proline uptake stimulating amino acids. It is concluded that the obligatory pathway of L-amino acid--D-amino acid--oxo acid which exists in the oxidation of L-alanine does not exist with other L-amino acids. It is likely that other D-amino acids in the pool are formed in the presence of D-amino acid oxidase or D-amino acid aminotransferase.

A new colorimetric determination of d-amino acid oxidase and urate oxidase activity

A new method of colorimetric determination of d-amino acid oxidase and urate oxidase using catalase and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole is reported. This method is based on the combination of two steps of enzyme reactions and colorimetric procedure. The values obtained by this method are satisfactorily correlated with those obtained by the dinitrophenylhydrazine method for d-amino acid oxidase activity and the ultraviolet method for urate oxidase activity and showed good reproducibility and accuracy. It is considered that the method can be useful as a method of activity determination for studying enzyme kinetics and the reaction mechanism.

Immobilization of yeast microbodies and the properties of immobilized microbody enzymes

Yeast microbodies isolated from methanol-grown cells of Kloeckera sp. No. 2201 were immobilized by two types of entrapping techniques: photocrosslinking of liquid oligomers of suitable photosensitive resins and crosslinking of albumin molecules with glutaraldehyde. The apparent activities of catalase, alcohol oxidase, and D-amino acid oxidase in the gel-entrapped microbodies were 40–50, 70–80, and ca. 50% respectively as compared with those in the free microbodies. Alcohol oxidase in the immobilized microbodies, similarly to that in free ones, oxidized methanol, ethanol, n-propanol, n-butanol, n-amyl alcohol, and benzyl alcohol. Some properties of catalase and alcohol oxidase in the microbodies immobilized by the above-mentioned techniques were studied in comparison with those of the enzymes in the free microbodies.

Evidence for the presence of two D-amino acid oxidases inPseudomonas aeruginosa PAO

By the isolation of mutants that were unable to grow on L-hydroxyproline or DL-valine, it has been possible to demonstrate the presence of two different types of D-amino acid oxidase activities inPseudomonas aeruginosa PAO. One was the D-amino acid dehydrogenase, probably involved in the oxidation of a number of D-amino acids such as D-alanine, D-phenylalanine and D-valine. The other was the inducible oxidase, specific to the oxidation of allohydroxy-D-proline formed from L-hydroxyproline during its oxidation. Thus, it has been possible to delink the involvement of the general D-amino acid dehydrogenase in the oxidative breakdown of allohydroxy-Dsproline.

Immobilization of yeast microbodies by inclusion with photo-crosslinkable resins.

Yeast microbodies containing FAD-dependent alcohol oxidase, catalase and D-amino acid oxidase were isolated from methanol-grown cells of Kloeckera sp. 2201 and immobilized intact in matrices formed by a short-time illumination of photo-crosslinkable resin oligomers. The relative activities of catalase, alcohol oxidase and D-amino acid oxidase of the gel-entrapped microbodies were 36, 76 and 31% respectively as compared with those of free microbodies. Immobilization enhance d the stability of catalase to a certain degree, but not that of alcohol oxidase. The pH/activity profiles of catalase and alcohol oxidase of the entrapped organelles showed more narrow pH optima than those of the free counterparts. D-Amino acid oxidase in immobilized microbodies showed a somewhat higher Km value for D-alanine than that in free ones. Immobilized microbodies oxidized two moles of methanol to form two moles of formaldehyde with consumption of one mole of molecular oxygen. Addition of 3-amino-1,2,4-triazole, an inhibitor of catalase, reduced the formation of formaldehyde to half the amount without change in the amount of oxygen consumed, indicating the synergic action of alcohol oxidase and catalase in methanol oxidation in the microbodies of living yeast cells.

Isolation and characterization of a mitochondrial D-amino acid oxidase from Neurospora crassa

D-Amino acid oxidase (EC 1.4.3.3) activity in homogenates of Neurospora crassa strain SY7A was found to sediment with the mitochondrial fraction. Digitonin fractionation studies on purified mitochondria have indicated a matrix localization of the enzyme. Additionally, a peroxidase (EC 1.11.1.7) activity, which may remove hydrogen peroxide formed as a product of D-amino acid oxidation, was also found in the mitochondrial matrix. Partial purification (20- to 30-fold) of the mitochondrial D-amino acid oxidase was achieved. The enzyme exhibited a pH optimum between 9.0 and 9.2, temperature optimum between 20 and 30 degrees C, and a molecular weight of 118 000 +/- 6000 as determined by gel electrophoresis and 125 000 as determined by gel chromatography.

Human lymphocyte response to d-valine media

Failure of human lymphoid cell lines to grow in d-valine-substituted media is associated with the lack of d-amino acid oxidase activity in these cells.

Studies on Peroxisomes. VII. Effect of the Light Component on the Formation of the Core-DAAO Complex of Rat Liver Peroxisomes

Partially purified peroxisomal core containing and not containing the light component was isolated from the liver homogenate of the rat and used for formation of the core complex bearing D-amino acid oxidase (DAAO). When either core preparation was vigorously stirred in the DAAO preparation at 0°C for 30 min and then centrifuged at 20, 000g for 30 min, the urate oxidase activity as a core marker was found in the pellet in which a small amount of DAAO activity was also found. The peroxisomal core containing the light component had a tendency to constitute the core-DAAO complex more easily than core without the component. The light component was extracted from the partially purified core (100-fold over the liver homogenate). The light component had a high cholesterol content. An experiment on reconstitution of the approximate native core complex using preparations of the light component, DAAO and the highly purified core (about 450-fold over the liver homogenate) showed that the peroxisomal core complex seemed to consist of urate oxidase, other peroxisomal oxidases, such as DAAO, and the light component, and also showed that DAAO and the light component probably underwent some interactions when binding to the core.

Isolation of peroxisomes from the dog kidney cortex

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, β-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and d-amino acid oxidase for peroxisomes. The activities of d-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while d-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and d-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.