Multiple amine oxidases in cucumber seedlings

Cell-free extracts of cucumber (Cucumis sativus L. cv. National Pickling) seedlings were found to have amine oxidase activity when assayed with tryptamine as a substrate. Studies of the effect of lowered pH on the extract indicated that this activity was heterogeneous, and three amine oxidases could be separated by ion exchange chromatography. The partially purified enzymes were tested for their activities with several substrates and for their sensitivities to various amine oxidase inhibitors. One of the enzymes may be a monoamine oxidase, although it is inhibited by some diamine oxidase inhibitors. The other two enzymes have properties more characteristic of the diamine oxidases. The possible relationship of the amine oxidases to indoleacetic acid biosynthesis in cucumber seedlings is discussed.     

Structural insights into the mechanism of amine oxidation by monoamine oxidases A and B

Due to their pharmacological importance in the oxidation of amine neurotransmitters, the membrane-bound flavoenzymes monoamine oxidase A and monoamine oxidase B have attracted numerous investigations and, as a result, two different mechanisms; the single electron transfer and the polar nucleophilic mechanisms, have been proposed to describe their catalytic mechanisms. This review compiles the recently available structural data on both enzymes with available mechanistic data as well as current NMR data on flavin systems to provide an integration of the approaches. These conclusions support the proposal that a polar nucleophilic mechanism for amine oxidation is the most consistent mechanistic scheme as compared with the single electron transfer mechanism.     

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.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Oxidative deamination of biogenic amines by intestinal amine oxidases: histamine is specifically inactivated by diamine oxidase.

The ability of the gut to inactivate various amines by oxidative deamination was tested with a 130-fold purified amine oxidase preparation from dog small intestine. Of 34 amines tested, putrescine, benzylamine, cadaverine, and serotonin were the most favourable substrates. Histamine was inactivated rapidly by this enzyme preparation, too. Histamine derivatives methylated at the imidazole nucleus were also deaminated, whereas Nalpha-methylhistamine was only a poor substrate and Nalpha, Nalpha-dimethylhistamine was not a substrate at all. Using a second procedure for the purification of amine oxidases from gut, the separation of a soluble monoamine oxidase from diamine oxidase was achieved by gel filtration on Sephadex G-200. The diamine oxidase deaminated putrescine (Km = 1.3 x 10(-4)M) and histamine (Km = 6.6 x 10(-5)M), but not serotonin, and was inhibited by aminoguanidine, but not by pargyline. The soluble monoamine oxidase inactivated serotonin (Km = 4.5 x 10(-4)M), but not histamine and putrescine and was inhibited by pargyline, but not by aminoguanidine. It was concluded that in dog small intestine (as well as in rabbit small intestine) only diamine oxidase was capable of inactivating histamine by oxidative deamination.     

Synthesis of 4-methyl-thio-phenyl-propylamine and the evaluation of its interaction with different amine oxidases

A new molecule, the 4-methyl-thio-phenyl-propylamine (PrNH(2)) was synthesized and its biological interaction with different amine oxidases such as semicarbazide sensitive amine oxidase (SSAO) [E.C.1.4.3.6], and monoamine oxidase [E.C.1.4.3.4] under its two isoforms, MAO A and MAO B, has been assessed. The substrate specifities of MAO and SSAO overlap to some extent. In this context, the search of new molecules, able to discriminate between these different amine oxidases is very important as it will allow greater elucidation of the SSAO's role in physiological and pathological conditions. We report for the first time, the synthesis and evaluation of a new molecule which has a high affinity towards the SSAO family of enzymes, more so than previously described and furthermore an ability to discriminate between the different amine oxidases.     

Screening of the occurrence of copper amine oxidases in Fabaceae plants

Aim of this work was to find the best source for obtaining high amount of copper amine oxidase (EC 1.4.3.6) that can be further used for analytical or industrial applications. The study focused on plant enzymes, because they occur in much higher content in the starting material than the enzymes from other sources, have higher specific activity and are also more thermostable. Presence of the amine oxidase was tested in extracts from 4 to 7-d-old seedlings of thirty-four various Fabaceae plants. Amine oxidases from nine selected plants were purified by general method involving ammonium sulfate fractionation, controlled heat denaturation, and three chromatographic steps. Kinetic properties of the amine oxidases purified were tested with a wide range of substrates and inhibitors and were found to be very similar. Best purification yield, and total and specific activities were obtained for the enzyme from grass pea (Lathyrus sativus) throughout all purification steps. Hence, the grass pea extract was chosen as a suitable candidate for massive production of the amine oxidase. This revised version was published online in July 2006 with corrections to the Cover Date.     

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.     

Mechanism-based inactivation of monoamine oxidases A and B by tetrahydropyridines and dihydropyridines.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its primary oxidation product, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), are mechanism-based inhibitors of monoamine oxidases A and B. The pseudo-first-order rate constants for inactivation were determined for various analogues of MPTP and MPDP+ and the concentrations in all redox states were measured throughout the reaction. Disproportionation was observed for all the dihydropyridiniums, but non-enzymic oxidation was insignificant. The dihydropyridiniums were poor substrates for monoamine oxidase A and, consequently, inactivated the enzyme only slowly, despite partition coefficients lower than those for the tetrahydropyridines. For monoamine oxidase B, the dihydropyridiniums were more effective inactivators than the tetrahydropyridines. Substitutions in the aromatic ring had no major effect on the inactivation of monoamine oxidase B, but the 2'-ethyl- and 3'-chloro-substituted compounds were very poor mechanism-based inactivators of monoamine oxidase A. It is clear that both oxidation steps can generate the reactive species responsible for inactivation.     

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.     

A sensitive photometric assay for monoamine oxidase.

A new spectrophotometric assay for the determination of monoamine oxidase activity is described. This simple and sensitive method is based on a coupled indicator reaction measuring the monoamine oxidase-dependent production of hydrogen peroxide. In this reaction the hydrogen peroxide-dependent oxidation of leuco-2′,7′-dichlorofluorescein to 2′,7′-dichlorofluorescein catalyzed by horseradish peroxidase is followed at 502 nm. Using benzylamine and seven biogenic amines as substrates, linear relationships between 2′,7′-dichlorofluorescein formation rate and monoamine oxidase concentration were found. The assay is especially suitable for determining substrate specificities for physiological amines as well as for inhibitor studies with pargyline or the monoamine oxidase A- and B-specific inhibitors clorgyline and deprenyl.     

Inhibition of amine oxidases activity by 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives

A novel series of 1-acetyl-3,5-diphenyl-4,5-dihydro-(1H)-pyrazole derivatives have been synthesised and investigated for the ability to inhibit selectively monoamine oxidases, swine kidney oxidase, and bovine serum amine oxidase. The newly synthesised compounds 1–6 proved to be reversible and non-competitive inhibitors of all types of the assayed amine oxidases. Compounds inhibit monoamine oxidases potently, displaying low I₅₀ values of particular interest. In particular 1-acetyl-3-(2,4-dihydroxyphenyl)-5-(3-methylphenyl)-4,5-dihydro-(1H)-pyrazole 6 showed to be a potent monoamine oxidase inhibitor with a K_i of about 10⁻⁸ M. Further insights in the theoretical evaluation of the possible interactions between the compounds and monoamine oxidase B have been developed through a computational approach.     

Inhibition of plant amine oxidases by a novel series of diamine derivatives

A series of N,N'-bis(2-pyridinylmethyl)diamines was synthesized and characterized for their inhibition effects towards plant copper-containing amine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.5.3.11), which mediate the catabolic regulation of cellular polyamines. Even though these enzymes catalyze related reactions and, among others, act upon two common substrates (spermidine and spermine), their molecular and kinetic properties are different. They also show a different spectrum of inhibitors. It is therefore of interest to look for compounds providing a dual inhibition (i.e. inhibiting both enzymes with the same inhibition potency), which would be useful in physiological studies involving modulations of polyamine catabolism. The synthesized diamine derivatives comprised from two to eight carbon atoms in the alkyl spacer chain. Kinetic measurements with pea (Pisum sativum) diamine oxidase and oat (Avena sativa) polyamine oxidase demonstrated reversible binding of the compounds at the active sites of the enzymes as they were almost exclusively competitive inhibitors with K(i) values ranging from 10(-5) to 10(-3)M. In case of oat polyamine oxidase, the K(i) values were significantly influenced by the number of methylene groups in the inhibitor molecule. The measured inhibition data are discussed with respect to enzyme structure. For that reason, the oat enzyme was analyzed by de novo peptide sequencing using mass spectrometry and shown to be homologous to polyamine oxidases from barley (isoform 1) and maize. We conclude that some of the studied N,N'-bis(2-pyridinylmethyl)diamines might have a potential to be starting structures in design of metabolic modulators targeted to both types of amine oxidases.     

An important lysine residue in copper/quinone-containing amine oxidases

The interaction of xenon with copper/6-hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediterranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. (129)Xe chemical shift provided evidence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the analyzed plant amine oxidases showed that the k(c) value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured K(m) value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the K(m) value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the k(c) value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramatically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity.     

A New Rapid and Sensitive Bioluminescence Assay for Monoamine Oxidase Activity

The in vivo luminescence of an aldehyde-requiring mutant of the luminous bacteria Vibrio harveyi (M42) increases dramatically upon the addition of long-chain aliphatic aldehydes (C8-C16). The intensity of this luminescence is linearly related to aldehyde concentration. This property was utilized for the determination of monoamine oxidase activity using n-octylamine and n-decylamine as substrates, which are converted by monoamine oxidase to n-octylaldehyde and n-decylaldehyde, respectively. The addition of the amine to a suspension containing rat liver mitochondria and M42 cells initiated a luminescence that was directly proportional to monoamine oxidase activity according to two parameters: (1) the rate of the initial increase in luminescence and (2) the final "steady-state" level of luminescence. The new assay has advantages of high sensitivity, rapidity, the possibility to perform discontinuous as well as continuous monitoring of monoamine oxidase activity, and applicability to turbid preparations.     

The biological functions of polyamine oxidation products by amine oxidases: Perspectives of clinical applications

Summary. The polyamines spermine, spermidine and putrescine are ubiquitous cell components. If they accumulate excessively within the cells, due either to very high extracellular concentrations or to deregulation of the systems which control polyamine homeostasis, they can induce toxic effects. These molecules are substrates of a class of enzymes that includes monoamine oxidases, diamine oxidases, polyamine oxidases and copper containing amine oxidases. Polyamine concentrations are high in growing tissues such as tumors. Amine oxidases are important because they contribute to regulate levels of mono- and polyamines. These enzymes catalyze the oxidative deamination of biogenic amines and polyamines to generate the reaction products H₂O₂ and aldehyde(s) that are able to induce cell death in several cultured human tumor cell lines. H₂O₂ generated by the oxidation reaction is able to cross the inner membrane of mitochondria and directly interact with endogenous molecules and structures, inducing an intense oxidative stress. Since amine oxidases are involved in many crucial physiopathological processes, investigations on their involvement in human diseases offer great opportunities to enter novel classes of therapeutic agents.     

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.     

A spectrophotometric method for determining the oxidative deamination of methylamine by the amine oxidases

Previously published studies on the oxidative deamination of methylamine by the amine oxidases have determined the formation of radioactively labeled formaldehyde from [(14)C]methylamine. The present work describes a coupled spectrophotometric assay, using formaldehyde dehydrogenase, for the continuous determination of the oxidative deamination of methylamine by semicarbazide-sensitive amine oxidase (SSAO) and its potential use for determining methylamine concentrations in plasma. In this assay, the formaldehyde produced by methylamine deamination is further oxidized to formate, with the reduction of NAD(+), by formaldehyde dehydrogenase. The NADH generated is monitored continuously at 340 nm. Interference from the presence of a rotenone-insensitive NADH oxidase activity in crude tissue homogenates and microsomal fractions can be minimized by pretreating samples with Triton X-100 or substituting NAD(+) by APAD(+) in the coupled assay. This relatively inexpensive and reproducible assay procedure avoids the use of radioactively labeled material.     

High-performance liquid chromatography of aldehydes and acids formed in monoamine oxidase-catalyzed reactions

A rapid, sensitive, and specific method for the determination of monoamine oxidase (MAO) activities toward different substrates is described. The assay is based on high-performance liquid chromatographic (HPLC) separation and electrochemical detection of the aldehyde or acid products. The aldehyde metabolic intermediates were observed to be quite stable in 0.1 N perchloric acid containing antioxidant and EDTA, and therefore can be used to measure the MAO activity of washed mitochondrial membrane and partially purified or purified MAO. Incomplete conversion of aldehyde to acid was observed when the amine substrates were incubated with the crude enzyme preparations. These aldehydes can be converted to corresponding acids by addition of yeast aldehyde dehydrogenase and beta-NAD and the acid can also be measured by HPLC-electrochemical detection. A deuterium isotope effect in the oxidation of p-[alpha,alpha-2H2]tyramine and [alpha,alpha-2H2]serotonin has been demonstrated by this method.     

Comparison of kinetic properties of amine oxidases from sainfoin and lentil and immunochemical characterization of copper/quinoprotein amine oxidases

Kinetic properties of novel amine oxidase isolated from sainfoin (Onobrychis viciifolia) were compared to those of typical plant amine oxidase (EC 1.4.3.6) from lentil (Lens culinaris). The amine oxidase from sainfoin was active toward substrates, such as 1,5-diaminopentane (cadaverine) with K(m) of 0.09 mM and 1,4-diaminobutane (putrescine) with K(m) of 0.24 mM. The maximum rate of oxidation for cadaverine at saturating concentration was 2.7 fold higher than that of putrescine. The amine oxidase from lentil had the maximum rate for putrescine comparable to the rate of sainfoin amine oxidase with the same substrate. Both amine oxidases, like other plant Cu-amine oxidases, were inhibited by substrate analogs (1,5-diamino-3-pentanone, 1,4-diamino-2-butanone and aminoguanidine), Cu2+ chelating agents (diethyltriamine, 1,10-phenanthroline, 8-hydroxyquinoline, 2,2'-bipyridyl, imidazole, sodium cyanide and sodium azide), some alkaloids (L-lobeline and cinchonine), some lathyrogens (beta-aminopropionitrile and aminoacetonitrile) and other inhibitors (benzamide oxime, acetone oxime, hydroxylamine and pargyline). Tested by Ouchterlony's double diffusion in agarose gel, polyclonal antibodies against the amine oxidase from sainfoin, pea and grass pea cross-reacted with amine oxidases from several other Fabaceae and from barley (Hordeum vulgare) of Poaceae, while amine oxidase from the filamentous fungus Aspergillus niger did not cross-react at all. However, using Western blotting after SDS-PAGE with rabbit polyclonal antibodies against the amine oxidase from Aspergillus niger, some degree of similarity of plant amine oxidases from sainfoin, pea, field pea, grass pea, fenugreek, common melilot, white sweetclover and Vicia panonica with the A. niger amine oxidase was confirmed.