Oxidation of N-alkyl and C-alkylputrescines by diamine oxidases

N-Methyl-, N-ethyl-, N-propyl- and N-butylputrescine were assayed as substrates of diamine oxidase from pea seedling and pig kidney. With the exception of N-methylputrescine they were found to be oxidized to the corresponding aminoaldehydes. 1-Methyl-, 2-methyl-, 1-ethyl- and 1-propylputrescine were oxidized by the oxidases at lower rates than the N-alkylderivatives. 1,3-Dimethylputrescine had negligible oxidation rates while 1,4-dimethylputrescine (2,5-diaminohexane) was not a substrate. The oxidation of putrescine by the kidney oxidase was inhibited by 1,4-dimethylputrescine, while the pea oxidase was strongly inhibited by the former as well as by 2-methylputrescine and 1,3-dimethylputrescine. Serum amine oxidase did not oxidize the substituted putrescines although several of the latter inhibited spermidine oxidation by this oxidase.     

Optimized preparation and determination of pea seedling diamine oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Optimized Preparation and Determination of Pea Seedling Diamine Oxidase

The level of diamine oxidase in pea seedling stems has been determined as a function of time after germination in both etiolated and non-etiolated plants. The maximum amount of enzyme per plant is obtained between 11 and 13 days. The amount of activity per gram of tissue appears to be proportional to the rate of growth. We describe an efficient method of isolation of pea seedling stem diamine oxidase from 12-day-old etiolated seedlings, a procedure that brings the enzyme to purity after a 97-fold purification. A new assay procedure for pea seedling diamine oxidase is detailed and compared to previously used methods. The kinetic parameters for three common substrates have also been determined. SDS-acrylamide gel electrophoresis, gel filtration chromatography and copper analyses have been used to determine that pea seedling diamine oxidase exists as a dimer of two apparently identical subunits, the dimer molecular weight being about 190,000. The isoelectric point of this enzyme was determined to be 6.5.     

Pyrroloquinoline quinone, a method for its isolation and identification by mass spectrometry.

Procedures for the unambiguous detection and for the isolation and mass spectrometric identification of pyrroloquinoline quinone (PQQ) are presented. The procedure involved acid hydrolysis of protein in the presence of phenylhydrazine and successive isolation and identification of the formed adduct using mass spectrometry. In HPLC the phenylhydrazone of PQQ gave many methylated products, of which the predominant compound was the pentamethylated derivative. After reaction of the phenylhydrazone derivative of PQQ (PHPQQ) with ammonia, a product was obtained which did not contain phenylhydrazine and which formed a pentamethylated derivative as the main methylation product. The HPLC profiles of the methylated products of PHPQQ and of its ammonia derivative were very characteristic and could be used for identification in addition to mass spectrometry. However, prolonged treatment of proteins with phenylhydrazine during hydrolysis can result in the formation of a material that resembles PQQ in some aspects of its behaviour. Thus, analysis by MS is essential for unambiguous identification. This analytical procedure was applied to pig plasma benzylamine oxidase, pig aorta lysyl oxidase, pig kidney diamine oxidase and bovine serum albumin with negative results. However, samples of pronase contained variable quantities of non-covalently bound PQQ: this can lead to erroneous identification of PQQ in enzyme after pronase digestion.     

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.     

Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination

The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling.     

Inhibition of six copper-containing amine oxidases by the antidepressant drug tranylcypromine

Potential inhibitory effects of the clinically utilized monoamine oxidase inhibitor tranylcypromine (TCP) on mammalian, plant, bacterial, and fungal copper-containing amine oxidases have been examined. The following enzymes have been investigated: human kidney diamine oxidase (HKAO), bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), and Pichia pastoris lysyl oxidase (PPLO). Only BPAO, EPAO, and AGAO were found to lose significant levels of activity when incubated with varying amounts of TCP. Inhibition of BPAO was completely reversible, with dialysis restoring full activity. TCP inhibition of AGAO was also found to be ultimately reversible; however, dialysis did not remove all bound compounds. Chemical displacement with either substrate or a substrate analogue successfully removed all bound TCP, indicating that this compound has a high affinity for the active site of AGAO. The notable lack of TCP inhibition on HKAO argues against the inhibition of diamine oxidase as a potential source for some of the deleterious side effects occurring in patients treated with this antidepressant. The marked differences observed in behavior among these enzymes speaks to the importance of intrinsic structural differences between the active sites of copper amine oxidases (CAO) which affect reactivity with a given inhibitor.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

On the nature of chromophore in pig kidney diamine oxidase.

The nature of the 500-nm chromophore in pig kidney diamine oxidase was investigated by absorption spectroscopy and fluorescence in the presence of various chelating or carbonyl-specific reagents. From the spectroscopic measurements the following conclusions can be drawn. First, the 500-nm absorption band is not due to copper, the reduction of which is not related to the disappearance of this band. Second, phenylhydrazine and cycloserine give rise, upon reaction with the enzyme, to absorptions very similar to those of a pyridoxal enzyme, aspartate aminotransferase. Third, these enzyme derivatives are unexpectedly non-fluorescent. Copper removal, obtained after prolonged incubation of cycloserine-treated enzyme in the presence of reducing and chelating agents, leads to a fluorescence similar to that of cycloserine-aspartate transminase. It is proposed that copper is coordinated to the postulated pyridoxal phosphate of diamine oxidase through the pyridine nitrogen.     

The amine oxidases of human placenta and pregnancy plasma

1. The purification of monoamine oxidase and diamine oxidase from normal human term placental tissue is described. 2. The properties of these enzymes are reported and compared with the properties of unpurified human pregnancy plasma. 3. This comparison shows that the amine oxidase of pregnancy plasma has properties corresponding to purified placental diamine oxidase, suggesting a placental origin for the plasma enzyme system. 4. Detailed kinetic study of the purified placental diamine oxidase suggests that it has a Ping Pong sequence, a mechanism of action and rate-limiting step similar to the diamine oxidase of pig kidney. 5. It is suggested that the enzyme system is important in protecting the foeto-placental unit from excesses of biogenic amines.     

The depression of the synthesis of pea diamine oxidase due to light and the verification of its participation in growth processes using competitive inhibitors

The time courses of the synthesis of diamine oxidase in pea plants grown for 14 days either in the light or in the dark are similar with the highest increase in activity occurring in the cotyledons and in the shoots during the first 6 to 8 days. Plants grown in the dark showed a 2- to 3-fold higher enzyme activity than plants grown in the light. Pea diamine oxidase could bein vivo efficiently inhibited by substrate analogues 1,4-diamino-2-butanone and 1,5-diamino-3-pentanone. The first compound inhibited proportionally to its concentration the growth of etiolated pea plants, but its instability makes an unequivocal interpretation of the results difficult. On the other hand, 1,5-diamino-3-pentanone a stable and more efficient diamine oxidase inhibitor depressed the growth of pea seedlings only at concentrations as high as 5 mM and 10 mM, at which the growth of cress seedlings not containing diamine oxidase was also strongly depressed. The results obtained indicate that tryptamine oxidation catalyzed by diamine oxidase is not involved in the main metabolic pathway leading from tryptophan to indoleacetate in pea plants.     

Stereospecific deamination of benzylamine catalyzed by different amine oxidases

1. Stereospecific deuterated benzylamine enantiomers, R(alpha-2H1)-and S(alpha-2H1)-benzylamine, were synthesized by a combined chemical and enzymatic method. 2. The retention or cleavage of the deuterium atom during deamination of benzylamine catalyzed by amine oxidases from different sources was assessed by a GC-MS procedure and confirmed by HPLC separation of the products and by the observation of a deuterium isotope effect. 3. Three types of stereospecific abstraction of hydrogen atoms from the alpha-carbon of benzylamine during deamination were observed: (a) In the first type of deamination the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial MAO from different tissues; (b) The second type of deamination involves the abstraction of pro-S hydrogen. 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; and (c) Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. 4. The kinetic deuterium isotope effect during the deamination of benzylamine by the different amine oxidase varies greatly, i.e. VH/VD ranged from 1.7 to 4.0.     

Involvement of superoxide in the catalytic cycle of diamine oxidase.

The reaction of pig kidney diamine oxidase (amine:oxygen oxidoreductase (deaminating) (pyridoxal-containing), EC 1.4.3.6) could be significantly inhibited by superoxide dismutase active copper chelates but not by native 2Cu,2Zn-superoxide dimutase (cuprein). The ligands alone as well as Cd2+, a heavy metal of similar toxicity to Cu2+, showed no inhibition whatsoever. This indicates that .O-2 participates in the catalytic cycle and is produced at a site scarcely accessible to such a large molecule as cuprein. A mechanism for the second, aerobic step of the diamine oxidase reaction is suggested.     

Reaction between mammalian amine oxidases and their antibodies

Antibodies have been raised against purified beef plasma, pig plasma and pig kidney amine oxidases. Despite the overall similarity, no immunological cross-reactivity was observed among these enzymes, even using a very sensitive light-scattering technique. The presence of substrate affects the rate of the reaction between kidney diamine oxidase and its antibody, but not that of other amine oxidases.     

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     

Stereochemical trends in copper amine oxidase reactions

Copper amine oxidases (EC 1.4.3.6) exhibit atypical stereochemical patterns in the reactions they catalyze. Dopamine and tyramine are oxidized with abstraction of the pro-R hydrogen by the porcine plasma amine oxidase, the pro-S hydrogen by pea seedling amine oxidase and a net nonstereospecific proton abstraction by the bovine plasma enzyme. This provides the first example in which a reaction catalyzed by enzymes in the same formal class occurs by all three possible stereochemical routes. To assess the underlying mechanistic significance of this heterogeneity, we have established the stereochemical course of the oxidation of tyramine by five additional copper amine oxidases using 1H NMR spectroscopy. Reactions catalyzed by rabbit and sheep serum amine oxidases are nonstereospecific. These enzymes exhibit rare mirror image binding with differential flux through two opposite and stereospecific reaction pathways. Differential primary kinetic isotope effects are observed for each mode, 8 and 4.6 for pro-S abstraction and 2.6 and 2.7 for pro-R abstraction by the sheep and rabbit amine oxidases, respectively. Tyramine oxidations catalyzed by the soybean and chick pea amine oxidases and porcine kidney diamine oxidase, however, are all stereospecific, occurring with loss of the pro-S hydrogen at C-1. Solvent exchange profiles are consistent within each stereochemical class of enzyme; the pro-R and nonstereospecific enzymes exchange solvent into C-2 of product aldehydes, the pro-S enzymes do not.     

Malondialdehyde production from spermine by homogenates of normal and transformed cells

The oxidation of spermine in vitro by a mixture of polyamine oxidase and diamine oxidase from pig kidney gives rise to malondialdehyde via 3-aminopropanol as the intermediate. Conversely, with spermidine, under similar experimental conditions, no evidence could be obtained for malondialdehyde formation within the limits of sensitivity of the assay (2.0 nmol). The activities of both these enzymes show about a 2-fold increase in normal rat kidney cells (LA31 NRK) transformed by the temperature sensitive mutant of Rous sarcoma virus (LA31) and incubated at the non permissive temperature (39 degrees C) compared to the activities in LA31 NRK at the permissive temperature (33 degrees C). These same enzymatic activities show no temperature dependent changes in normal rat kidney cells (NRK) or in these same cells infected by the wild type virus (NRK B77). In extracts derived from Friend erythroleukemic cells induced to differentiate by dimethyl sulfoxide or hexamethylene bis acetamide, spermine oxidation takes place more efficiently than in non induced cells. A rise in diamine oxidase activity is seen in LA31 NRK (39 degrees C) 12 h after the temperature shift, whereas morphological manifestations of normalcy are seen only at 48 h. The Km of diamine oxidase is 10(-6) M for putrescine and 10(-3) M for 3-aminopropanol. A possible mechanism involving the well documented acetylation of putrescine [23,26] is proposed for diverting intracellular putrescine away from cytosolic diamine oxidase and towards intramitochondrial monoamine oxidase.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.     

Monoamine and diamine oxidative deamination in the longitudinal smooth muscle of guinea pig ileum

The longitudinal smooth muscle of guinea pig ileum contains three different types of oxidative deaminating enzymes: monoamine oxidase types A and B, diamine oxidase and a soluble clorgyline-deprenyl-resistant benzylamine oxidase. These enzymes have different subcellular locations. The longitudinal smooth muscle of guinea pig ileum oxidatively deaminates beta-phenylethylamine at a much higher rate than benzylamine. beta-Phenylethylamine is a good substrate for monoamine oxidase type B but also for the soluble clorgyline-deprenyl-resistant benzylamine oxidase. On the other hand, benzylamine is oxidised by mitochondrial monoamine oxidase, by the clorgyline-deprenyl-resistant enzyme and by diamine oxidase.     

Regulation of diamine oxidase expression by beta 2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the beta 2-adrenergic mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, beta 1, beta 2- or beta 2, but not alpha 1-, alpha 2-, or beta 1-receptor-blocking agents prevented this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (alpha 1, alpha 2, beta 1, beta 2), isoproterenol (beta 1, beta 2) or terbutaline (beta 2) showed that also in normal rat kidney diamine oxidase activity is under the control of catecholamine-beta 2-receptors through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered beta 2-receptors coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.