Kinetic properties of rat kidney D-amino acid oxidase associated with peroxisomes

In contrast to hog kidney D-amino acid oxidase, the v vs s plots of D-amino acid oxidase in homogenized rat kidney did not have the form of a rectangular hyperbola, and showed an apparent negative cooperativity. After subcellular fractionation of rat kidney, both of the oxidases in the supernatant fraction and the peroxisomal fraction showed Michaelis-Menten type kinetics. The Km values for D-alanine and D-proline of the peroxisomal fraction were significantly lower than those of the supernatant fraction. The partially purified enzyme from the peroxisomal fraction showed the same kinetic properties as the supernatant fraction. These facts suggest that the two types of rat kidney D-amino acid oxidase were originally identical and that some interaction between the enzyme and peroxisomes is physiologically important for the function of the enzyme.     

Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.     

Avian and canine aldehyde oxidases. Novel insights into the biology and evolution of molybdo-flavoenzymes

Aldehyde oxidases are molybdo-flavoenzymes structurally related to xanthine oxidoreductase. They catalyze the oxidation of aldehydes or N-heterocycles of physiological, pharmacological, and toxicological relevance. Rodents are characterized by four aldehyde oxidases as follows: AOX1 and aldehyde oxidase homologs 1-3 (AOH1, AOH2, and AOH3). Humans synthesize a single functional aldehyde oxidase, AOX1. Here we define the structure and the characteristics of the aldehyde oxidase genes and proteins in chicken and dog. The avian genome contains two aldehyde oxidase genes, AOX1 and AOH, mapping to chromosome 7. AOX1 and AOH are structurally very similar and code for proteins whose sequence was deduced from the corresponding cDNAs. AOX1 is the ortholog of the same gene in mammals, whereas AOH represents the likely ancestor of rodent AOH1, AOH2, and AOH3. The dog genome is endowed with two structurally conserved and active aldehyde oxidases clustering on chromosome 37. Cloning of the corresponding cDNAs and tissue distribution studies demonstrate that they are the orthologs of rodent AOH2 and AOH3. The vestiges of dog AOX1 and AOH1 are recognizable upstream of AOH2 and AOH3 on the same chromosome. Comparison of the complement and the structure of the aldehyde oxidase and xanthine oxidoreductase genes in vertebrates and other animal species indicates that they evolved through a series of duplication and inactivation events. Purification of the chicken AOX1 protein to homogeneity from kidney demonstrates that the enzyme possesses retinaldehyde oxidase activity. Unlike humans and most other mammals, dog and chicken are devoid of liver aldehyde oxidase activity.     

d-ASPARTATE OXIDASE ACTIVITY IN EXTRACTS OF MAMMALIAN CENTRAL NERVOUS TISSUE

Abstract— d -Aspartate oxidase activity has been measured in water extracts of acetone powders prepared from cat forebrain, cerebellum and spinal cord, rat brain, hog brain and sheep brain stem, and compared with that found in rabbit and cat kidney. The results suggest that the brain enzyme has very similar properties to the n-aspartate oxidase ( d -aspartate: oxygen oxidorcductase (deaminating), EC 1.4.3.1) of kidney. Crude extracts (ammonium sulphate fractions of water extracts of acetone powders) displayed little activity without added FAD. FMN could not replace FAD. With oxygen as electron acceptor, the enzyme oxidized d -aspartate much more rapidly than d -glutamate, and displayed quite high activities with N -substituted derivatives of d -aspartate as substrates. Those amino acids susceptible to oxidation by d -amino acid oxidase were not oxidized by the d -aspartate oxidase. The regional distribution of the d -aspartate oxidase activity within the CNS differed from that of d -amino acid oxidase. As has been previously observed for kidney d -aspartate oxidase activity, dicarboxylic acids competitively inhibited this enzymic activity in brain extracts, while sodium benzoate and sodium barbitone, inhibitors of d -amino acid oxidase, were without effect.     

Isolation, purification, and properties of boar sperm cytochrome oxidase

Cytochrome oxidase was isolated from the midpiece of boar sperm by extraction and fractionation with ammonium sulfate in the presence of cholate. The enzyme was further purified to apparent homogeneity by DEAE-cellulose column chromatography in the presence of minimal amounts of Triton X-100. The purified enzyme exhibited similar oxidized and reduced optical spectra to those of the bovine heart and rat liver cytochrome oxidases. However, the sperm oxidase was found to contain stoichiometrically more subunits I, II, and III than the other, smaller subunits. The sperm oxidase also required phospholipids for its activity, but it was less sensitive to inhibition by KCN. Interestingly, the sperm oxidase was much more acid-stable than the bovine heart and rat liver counterparts. The optimumpH for the sperm oxidase catalyzing the electron transfer between ferrocytochromec and cytochromea was aroundpH 4.8, and those for the bovine heart and rat liver were 6.2 and 6.8, respectively. AtpH 4.5, the sperm oxidase still maintained about 70% enzyme activity, whereas less than 20% activity remained in the heart and liver oxidases. The peculiar properties of sperm cytochrome oxidase may be due to the fact that the well-packed chromosomes in the sperm headpiece do not function, such that the nuclear gene-coded subunits are deficient in the sperm cytochrome oxidase. The finding that the sperm oxidase was more acid-stable is an example of structure-function coordination, and is discussed from the viewpoint of chemiosmotic theory and the unique structure and functions of the sperm mitochondria.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

THE DEVELOPMENT OF D-AMINO ACID OXIDASE IN RAT CEREBELLUM

D-Amino acid oxidase (D-amino acid: O₂ oxidoreductase (deaminating), EC 1.4.3.3; D-AAO) activity is biochemically undetected in rat brain stem, cerebellum and forebrain until 14 days after birth. Adult levels are attained by day 30 in the brain stem, and by day 36 in the cerebellum. At adulthood, forebrain D-AAO activity per g wet weight of tissue is less than 2% that of the cerebellum. In contrast to the pattern in the CNS, substantial D-AAO activity is present in both liver and kidney 2 days before birth and adult levels are approached within 2 weeks of birth. Nonetheless, D-AAO activities in rat liver, kidney, brain stem and cerebellum are likely to be due to a single enzyme which has properties very similar to the purified hog D-AAO. The late ontogenesis of D-AAO activity in cerebellum and brain stem relative to that in liver and kidney parallels reported phylogenetic data. Histochemical staining for D-AAO in rat cerebellar cortex is absent until 15 days after birth when activity is first observed in some cells of the external germinal zone and adjacent molecular layer. These cells appear to migrate to a final destination around the Purkinje cell soma and leave processes at the pial surface. By 21 days of age an adult pattern of staining is manifest throughout the cerebellum but it is of weak intensity. The adult pattern includes some staining in the granular layer which seems to be associated with mossy fibers and certain cerebellar glomeruli, and strong staining at the pial surface, in the molecular layer, and in cells surrounding, but not within, the Purkinje cell soma. The data suggest that the biochemical appearance of D-AAO in developing cerebellum derives from two sources: one associated with differentiation of one of the last cell types to form from the external germinal zone, and the other with maturation of mossy fibers and their synapses (cerebellar glomeruli).     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Species-dependent differences of the biochemical properties of diamine oxidase

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.     

Androgenic sensitivity of polyamine-oxidizing enzyme activities in female rat tissues

Summary. Effects of testosterone (10 μg/100 g body weight) on polyamine-oxidizing enzyme activities in female rat uterus, liver and kidney were demonstrated. Testosterone-treated rats exhibited 2.07 fold (p < 0.002) higher uterine polyamine oxidase (PAO) activity and 1.93 fold (p < 0.02) higher diamine oxidase (DAO) activity, as compared to the controls. In the liver, testosterone caused an elevation in PAO (1.39 fold, p < 0.05), but not in DAO activity, whereas in kidney the hormone stimulated DAO (1.30 fold, p < 0.05), but not PAO activity. The effects observed suggest a possible role for testosterone in the modulation of polyamine levels in the female organs studied and especially in uterus. Received May 12, 1999, Accepted December 16, 1999     

Presence of D-amino-acid oxidase protein in mutant mice lacking D-amino-acid oxidase activity.

1. Mutant mice lacking D-amino-acid oxidase activity were examined as to whether they possessed the enzyme protein. 2. Immunoblotting using an antibody against hog kidney D-amino-acid oxidase showed that kidney homogenates of the mutant mice as well as that of the normal mice had proteins reactive to the antibody. 3. Peroxisomal proteins of the kidney cells of the mutant mice were not different from those of the normal mice. 4. The peroxisomes of the mutant mice possessed a protein reactive to the antibody in the immunoblotting whose size was the same as the D-amino-acid oxidase protein present in the peroxisomes of the normal mice. 5. These results suggest that the mutant mice synthesize the D-amino-acid oxidase protein and integrate it into peroxisomes, though it is a nonfunctional enzyme.     

D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes.

D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases.     

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

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. All enzyme activities were higher at alkaline than at neutral pH in both groups. 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 decrease 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. The differences suggest that the changes of peroxisomal enzyme activities by DEHP treatment are not directly related to peroxisome proliferation. 5. DEHP treatment caused a marked increase of total and peroxisomal fatty acid oxidation in rat liver homogenates. 6. In the control group the rate of peroxisomal fatty acid oxidation was higher at alkaline pH than at neutral pH. 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 beta-oxidation.     

Unusual responses of rat hepatic and renal peroxisomes to RMI 14, 514, a new hypolipidemic agent.

RMI 14, 514 ([5-tetradecycloxy]-2-furancarboxylic acid) represents a new class of hypolipidemic agents which cause unusual ultrastructural changes in liver of male rats and in selected peroxisomal enzymes in liver and kidney of both sexes. Among the principal ultrastructural changes in peroxisomes of male rat liver were (a) cavitation and compartmentalization of the matrix, often giving the appearance of a peroxisome-within-a-peroxisome, and (b) narrow, dense extensions of canaliculi or cisterns from the periphery of the peroxisome, forming partial circlets or surrounding irregular areas of cytoplasm. The unusual enzyme responses were (a) elevation of catalase activity in liver and kidney in female rats, (b) increased activity of three hydrogen peroxide-producing oxidases (urate oxidase, L-alpha-hydroxy acid oxidase, and D-amino acid oxidase) in the liver of both sexes, and (c) elevation of activity of the last two oxidases in male kidney. The peculiar ultrastructural changes in liver peroxisomes combined with the responses of selected peroxisomal enzymes represent unusual modulations or adaptations of these organelles to a hypolipidemic agent, the effects of which have not been reported extensively.     

Ornithine decarboxylase, transglutaminase, diamine oxidase and total diamines and polyamines in maternal liver and kidney throughout rat pregnancy.

Ornithine decarboxylase (ODC; EC 4.1.1.17), transglutaminase (EC 2.3.2.13), diamine oxidase (DAO; EC 1.4.3.6) and total di- and poly-amines were studied in rat liver and kidney cortex throughout pregnancy. In liver, ODC activity exhibited two major peaks (4.5-5 times the control activities) on days 15 and 17. Also putrescine and spermidine increased biphasically (3-4-fold), but no variation in spermine content was observed. Transglutaminase activity showed slight variations only near the end of gestation. In kidney, ODC activity did not fluctuate significantly during pregnancy, whereas both transglutaminase activity and putrescine content showed three major increases, in very early, middle and late pregnancy. No significant variations in spermidine and spermine were observed. In both organs, DAO activity, very low or undetectable until day 10, dramatically increased (10- and 20-fold in kidney and liver respectively) in the second half of pregnancy, reaching maxima on days 16-17 and 19. The results obtained for transglutaminase, ODC and total di- and poly-amines are interpreted on the basis of hyperplastic and hypertrophic events in the liver and kidney respectively. The behaviour of DAO suggests that the enzyme plays an important role in the control of intracellular diamine concentration.     

Possible influence of gonadotrophins on formation in vivo of pyridoxal phosphate

FSH administered to normal rats increased the activity of pyridoxine phosphate oxidase of both liver and kidney and, consequently, pyridoxal phosphate levels in these tissues were elevated. LH administration, on the other hand, decreased the activity of pyridoxine phosphate oxidase, resulting in diminished pyridoxal phosphate level in the tissues. The stimulatory effect of FSH on the activity of liver and kidney pyridoxine phosphate oxidase was not observed in castrated-adrenalectomised rats unless supplemented with cortisone and testosterone, respectively. Puromycin treatment prevented the FSH-induced rise in the activity of liver and kidney pyridoxine phosphate oxidases. It is suggested that FSH stimulates the activity of liver and kidney pyridoxine phosphate oxidase by increasing the synthesis of apoproteins of the enzyme, and the effect of FSH on liver is dependent on the presence of adrenal corticoids while the presence of testosterone is a prerequisite for the FSH to have its effect on kidney pyridoxine phosphate oxidase.     

Demonstration of an Antigenic Relationship Between Hog Cholera and Bovine Viral Diarrhea Viruses by Immunofluorescence

Specific staining of antigen within bovine embryo kidney tissue culture cells, infected with either Oregon C24V or NADL-MD bovine viral diarrhea virus, was accomplished using fluorescein-conjugated swine anti-hog cholera or bovine antiviral diarrhea globulin. Also specific staining of antigen within pig kidney tissue culture cells, infected with hog cholera virus, was accomplished using the same two types of conjugates. Specificity was confirmed by appropriate controls. The authors found immunofluorescence to be a convenient and sensitive method for determining an antigenic relationship between hog cholera and bovine viral diarrhea viruses.     

Separate peroxisomal oxidases for fatty acyl-CoAs and trihydroxycoprostanoyl-CoA in human liver

Fatty acyl-CoAs as well as the CoA esters of the bile acid intermediates di- and trihydroxycoprostanic acids are beta-oxidized in peroxisomes. The first reaction of peroxisomal beta-oxidation is catalyzed by acyl-CoA oxidase. We recently described the presence of two fatty acyl-CoA oxidases plus a trihydroxycoprostanoyl-CoA oxidase in rat liver peroxisomes (Schepers, L., P. P. Van Veldhoven, M. Casteels, H. J. Eyssen, and G. P. Mannaerts. 1990. J. Biol. Chem. 265: 5242-5246). We have now developed methods for the measurement of palmitoyl-CoA oxidase and trihydroxycoprostanoyl-CoA oxidase in human liver. The activities were measured in livers from controls and from three patients with peroxisomopathies. In addition, the oxidase activities were partially purified from control livers by ammonium sulfate fractionation and heat treatment, and the partially purified enzyme preparation was subjected to chromatofocusing, hydroxylapatite chromatography, and gel filtration. In earlier experiments this allowed for the separation of the three rat liver oxidases. The results show that human liver, as rat liver, contains a separate trihydroxycoprostanoyl-CoA oxidase. In contrast to the situation in rat liver, no conclusive evidence was obtained for the presence of two fatty acyl-CoA oxidases in human liver. Our results explain why bile acid metabolism is normal in acyl-CoA oxidase deficiency, despite a severely disturbed peroxisomal fatty acid oxidation and perhaps also why, in a number of other cases of peroxisomopathy, di- and trihydroxycoprostanic acids are excreted despite a normal peroxisomal fatty acid metabolism.