Occurrence of diamine oxidase in the apoplast of pea epicotyls

Most of the diamine oxidase (EC 1.4.3.6) present in pea (Pisum sativum L. cv. Rondo) epicotyls is found in the fluid obtained by centrifuging pea epicotyl sections previously infiltrated under vacuum with a buffer solution. No detectable amount of the cytoplasmic enzyme glucose-6-phosphate dehydrogenase is present in this fluid, showing that there is very little contamination by cell contents. Polyacrylamide-gel electrophoresis and specific-activity data indicate that diamine oxidase is the most plentiful protein in the extracellular solution obtained from pea epicotyl sections and that an active process is involved in the selective transfer of the enzyme outside the cell. The possible involvement of diamine oxidase in the supply of H₂O₂ to peroxidase-catalyzed reactions occurring inside the cell wall is discussed.Abbreviations DAO diamine oxidase - Glc6P glucose-6-phosphate     

Determination of the cross-points of rat liver peroxisomes,peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.     

Streptomyces sp. Z-11-6--a new producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254 Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep-mutagenesis and selection procedure allowed a genetically stable strain, Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Light inhibition of respiration is due to a dual function of the cytochrome b6-f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa

Studies of the respiratory electron transport pathway in the blue-green alga, Aphanocapsa, demonstrated the presence of cytochrome oxidase and a cytochrome complex. The use of antimycin A showed only the occurrence of a plastidal type of cytochrome complex (the cytochrome b6-f complex), which is insensitive to this inhibitor. Determination of the extent of photooxidation of cytochromes c-553 and f-556 under conditions of high and low cytochrome oxidase activities indicated an electron flow through both cytochromes to cytochrome oxidase. Direct evidence for a common segment of photosynthetic and respiratory electron transport from plastoquinone via the cytochrome b6-f complex to the soluble plastocyanin/cytochrome c-553 pool, as well as a competition between cytochrome oxidase and Photosystem I for reductants in this pool in the light, was obtained by measurements of electron transport with suitable electron donors in this alga.     

Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K _d 6.7 × 10^–7 M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavinbinding site of the enzyme, apo-L-aspartate oxidase has been reacted with N⁶-(6-carboxyhexyl)-FAD Succinimidoester. The structural characterization of the resulting N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/ monomer. Residue Lys38 was identified as the target of the covalent modification. N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.     

Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep mutagenesis-selection procedure allowed a genetically stable strain,Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria

Plastocyanin and cytochrome c6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b6f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time-resolved analysis showing that both cytochrome c6 and plastocyanin can be efficiently oxidized by the aa3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s(-1) for cytochrome c6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.     

6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans. Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase

The nucleotide sequence of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene of Arthrobacter oxidans is presented. This covalently flavinylated enzyme specifically oxidizes 6-hydroxy-D-nicotine to 6-hydroxy-N-methylmyosmine. Coinduced in the presence of nicotine is a 6-hydroxy-L-nicotine-specific enzyme, 6-hydroxy-L-nicotine oxidase (6-HLNO), with FAD noncovalently bound to the apoprotein. A comparison of the nucleotide-derived amino acid sequence of the 6-HDNO with the amino acid sequence data obtained from the purified 6-HLNO polypeptide suggests that the two enantiozymes expressed within the same cell are genetically unrelated. This conclusion is supported by the finding that the FAD-binding sites of the two enzymes are different. 6-HLNO exhibits at the amino-terminus of the polypeptide chain a dinucleotide-binding site characteristic for many other FAD- and NAD(P)-dependent enzymes. No such sequence was found in the nucleotide-derived amino acid sequence of 6-HDNO.     

Biodegradation of cyclohexylamine by Brevibacterium oxydans IH-35A

A bacterial strain capable of growing on cyclohexylamine (CHAM) was isolated by using enrichment and isolation techniques. The strain isolated, strain IH-35A, was classified as a member of the genus Brevibacterium. The results of growth and enzyme studies are consistent with degradation of CHAM via cyclohexanone (CHnone), 6-hexanolactone, 6-hydroxyhexanoate, and adipate. Cell extracts obtained from this strain grown on CHAM contained CHAM oxidase, and the model for CHAM oxidation by this enzyme was similar to the model for deamino oxidation of amine by amine oxidase.     

Extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6: preparation of it and its properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain, Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6-1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase from Streptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1-0.5% promoted the secretion of the extracellular glutamate oxidase.     

The critical relationship between free radicals and degrees of ischemia: evidence for tissue intolerance of marginal perfusion

Skin-flap ischemia has been associated with the presence of free radicals. In this study, two enzyme systems involved in free-radical metabolism were used to compare a distal skin flap to a skin graft. Forty-two rats were divided into several test groups. A 10 X 3 cm dorsal rat flap was used, and tissue biopsies for xanthine oxidase and malonyldialdehyde (MDA) were obtained 2.5, 5.5, and 8.5 cm from the base of the flap at the hours given. In group I (control), the flap was outlined but not elevated, and biopsies were obtained. In group II, the flap was elevated, and biopsies were obtained at 6 hours. In group III, the flap was elevated, the distal 4 X 3 cm was amputated and replaced as a full-thickness skin graft, and biopsies were obtained at 6 hours. In group IV, the flap was elevated, and biopsies were obtained at 12 hours. In group V, the flap was treated as in group III, and biopsies were obtained at 12 hours. In group VI, the flap was elevated, and biopsies were obtained at 24 hours. In group VII, the flap was treated as in group III, and biopsies were obtained at 24 hours. Results: Xanthine oxidase was significantly higher in all distal biopsies compared to proximal biopsies. Xanthine oxidase also increased with time. Malonyldialdehyde increased over time as well as with distance from the flap base. Distal flap biopsies at 24 hours had greatly increased levels of malonyldialdehyde compared to skin grafts (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

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, beta-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.     

The isolation of an o-diphenal oxidase from third-instar larvae of the blowfly Calliphora erythrocephala.

1. The isolation of an o-diphenol oxidase from an acetone-dried powder of late-third-instar larvae of Calliphora erythrocephala was investigated. An insoluble and micro-crystalline fraction containing the enzyme activity was obtained after fractionating extracts of the acetone-dried powder with (NH4)2SO4 and acetone. 2. This fraction can be solubilized in 0.1% sodium dodecyl sulphate without loss of activity. 3. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate shows that the o-diphenol oxidase is a minor component of the extracts from the acetone-dried powder. 4. The o-diphenol oxidase was purified by zonal centrifugation on a sucrose density gradient in the presence of sodium dodecyl sulphate. 5. The amino acid composition of the purified enzyme resembles that of some other o-diphenol oxidases. 6. The subunit composition of the o-diphenol oxidase is discussed.     

Protective effect of taurine on respiratory burst activity of polymorphonuclear leukocytes in endotoxemia

Summary. The aim of this study was to evaluate the effect of endotoxin on PMN leukocyte respiratory burst activity by measuring G6PD, NADPH oxidase and XO activities in guinea pig. In addition, the possible protective role of taurine against endotoxin-mediated PMN leukocyte function was examined. All experiments were performed with four groups (control, taurine, endotoxemia, taurine plus endotoxin) of ten guinea pigs. After the endotoxin was administrated (4 mg/kg) both G6PD and NADPH oxidase activities were significantly reduced compared with the control group. NADPH oxidase activity returned to the control value and G6PD activity also increased but it did not reach the control value. However when taurine was administrated (300 mg/kg) the activity of NADPH oxidase reached the control value; furthermore, G6PD activity also increased but it could not reach to the control value. When taurine was administrated alone, no effect on these enzymes was observed. Following the endotoxin administration, the activity of XO considerably increased. When taurine was administrated together with endotoxine and alone, this activity decreased compared to control value in both conditions. These results indicate that the O₂ ^•− formation in PMN leukocytes after the endotoxin administration is ensured by the catalysis of XO due to the inhibited NADPH oxidase activity. It was observed that taurine has considerable anti-inflammatory and antioxidant effects. However, conflicting results were obtained when taurine was administrated alone or together with an oxidant agent.     

The inactivation of pea-seedling diamine oxidase by peroxidase and 1,5-diaminopentane

1. The rate of oxidative deamination of 1,5-diaminopentane by pea-seedling extracts, which contain diamine oxidase [diamine-oxygen oxidoreductase (deaminating), EC 1.4.3.6], was increased by adding pyridoxal or pyridoxal phosphate. 2. Evidence was obtained that pyridoxal does not activate the apoenzyme of diamine oxidase, but prevents the inactivation of the enzyme. 3. This inactivation only occurred when 1,5-diaminopentane was the substrate and depended on a second thermolabile factor in the extract besides the diamine oxidase. 4. Purified diamine oxidase, when catalysing the oxidation of 1,5-diaminopentane, was rapidly inactivated in the presence of peroxidase. 5. The inactivation was prevented not only by pyridoxal and pyridoxal phosphate but also by several unrelated compounds including alpha-oxoglutarate, catechol and o-aminobenzaldehyde. 6. It is suggested that peroxidase catalyses the further oxidation of the product of the oxidative deamination of 1,5-diaminopentane to a compound that inactivates diamine oxidase. 7. The results diminish the relevance of previous evidence that plant diamine oxidase contains pyridoxal phosphate.     

Use of purified amyloglucosidase for the specific determination of total carbohydrate content of rat liver homogenate in a single step.

In prior studies, we employed a crude amyloglucosidase preparation, in conjunction with glucose oxidase reagent, to determine total carbohydrate in liver and muscle homogenates by a two-step procedure. Glycogen content was determined by subtracting tissue glucose (determined separately). By use of a purified amyloglucosidase, we have now verified the accuracy of two-step assay of rat liver homogenate with crude amyloglucosidase. The reliability of glucose oxidase detection of glucose in amyloglucosidase hydrolysates was established by comparing results with those obtained with hexokinase/glucose 6-phosphate dehydrogenase reagent. The availability of purified amyloglucosidase made it feasible to assay total carbohydrate content of homogenate in a single step, by incubating aliquots in the presence of both amyloglucosidase and glucose oxidase reagents. Results obtained with this one-step assay agreed with those of two-step analysis. To our knowledge, this is the first report of direct enzymic assay of total carbohydrate in rat liver homogenate, in a single step.     

Metabolism of pipecolic acid in a Pseudomonas species. V. Pipecolate oxidase and dehydrogenase

Oxidation of pipecolate to Delta(1)-piperideine-6-carboxylate is catalyzed by pipecolate oxidase, an inducible, membrane-bound dehydrogenase associated with the electron transport components of Pseudomonas putida P2. From the oxidase, we obtained a smaller particle containing flavine adenine dinucleotide (FAD) and cytochrome b, but no longer able to catalyze electron transfer to oxygen or to cytochrome c. Certain properties of this l-pipecolate dehydrogenase, an FAD-flavoprotein, are reported.