Absence of tissue-bound semicarbazide-sensitive amine oxidase activity in carp tissues

We have previously reported that carp (Cyprinus carpio) tissue mitochondria contain a novel form of monoamine oxidase (MAO), which belongs neither to MAO-A nor to MAO-B of the mammalian enzyme. This conclusion results from the findings that the carp MAO was equally sensitive to a selective MAO-A inhibitor clorgyline and to the MAO-B selective inhibitor l-deprenyl, when tyramine, a substrate for both forms, serotonin or beta-phenylethylamine, a substrate for either A or B-form of mammalian MAO, was used. In the present study, we tried to detect another amine oxidase, termed tissue-bound semicarbazide-sensitive amine oxidase (SSAO), activity in carp tissues. As definition of SSAO was used, such as insensitivity to inhibition of the kynuramine oxidizing activity by an MAO inhibitor pargyline and high sensitivity to the SSAO inhibitor semicarbazide. The results indicated that the oxidizing activity was selectively and almost completely inhibited by 0.1 mM pargyline alone or a combination of 0.1 mM pargyline plus 0.1 mM semicarbazide, but not by 0.1 mM semicarbazide alone. We also tried to detect any SSAO activity by changing experimental conditions, such as lower incubation temperature, higher enzyme protein concentration, a lower substrate concentration and different pH's in the reaction, as the enzyme source. However, still no SSAO activity could be detected in the tissues. These results conclusively indicate that carp tissues so far examined do not contain SSAO activity.     

Diabetes and semicarbazide-sensitive amine oxidase (SSAO) activity: a review

The enzyme of semicarbazide-sensitive amine oxidase (SSAO) activity has been reported to be elevated in blood from diabetic patients. SSAO are widely distributed in plasma membranes of various tissues and blood plasma. SSAO-mediated production of toxic aldehydes has been proposed to be related to pathophysiological conditions. Cytotoxic metabolites by SSAO may cause endothelial injury and subsequently induce atherosclerosis. The precise physiological functions of SSAO could play an important role in the control of energy balance in adipose tissue. It is possible that the increased SSAO activity in diabetes may be a result of up-regulation due to increase of SSAO substrates, such as methylamine or aminoacetone. SSAO could play an important role in the regulation of adipocyte homeostasis. Inhibition of SSAO could be of therapeutic value for treatment of diabetic patient.     

Interaction of bovine serum amine oxidase with the polyamine oxidase inactivator MDL 72527

MDL 72527 was considered a selective inhibitor of FAD-dependent polyamine oxidases. In the present communication, we demonstrate that MDL 72527 inactivates bovine serum amine oxidase, a copper-containing, TPQ-enzyme, time-dependently at 25 degrees C. In striking contrast, the enzyme remained active after incubation with excessive MDL 72527 at 37 degrees C, even after 70 h of incubation. Inactivation of BSAO with MDL 72527 at 25 degrees C did not involve the cofactor, as was shown by spectroscopy and by reaction with phenylhydrazine. Docking of MDL 72527 is difficult, owing to its size and two lipophilic moieties, and it has been shown that minor changes in reaction rate of substrates cause major changes in K(m) and k(cat)/K(m). We hypothesise that subtle conformational changes between 25 and 37 degrees C impair MDL 72527 from productive binding and prevent the nucleophilic group from reacting with the double bond system.     

Molecular characteristics of tissue-bound semicarbazide-sensitive amine oxidase (SSAO) in guinea pig tissues

Various mammalian tissues contain a tissue-bound amine oxidizing enzyme distinct from mitochondrial outer membrane enzyme, monoamine oxidase (MAO, EC 1.4.3.4), termed semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6). An increase in SSAO activity was found in patients suffering from vascular disorders such as diabetes and diabetic complications. It has previously been shown that 2-bromoethylamine (2-BEA) is a potent, and selective suicidal inhibitor of tissue-bound SSAO. The aim of this study was to investigate the interaction of this suicidal SSAO inhibitor with the tissue-bound enzyme in guinea pig lung, kidney, stomach, and heart homogenates. The conditions necessary for this inhibitor to titrate the concentrations of this enzyme were also determined. 2-BEA appears to interact with SSAO, as reported previously for this enzyme from different sources, in a manner consistent with an irreversible, "suicide" reaction. Because of this property, 2-BEA could be used to titrate the concentrations of SSAO active centers in these tissues under the appropriate conditions employed. Although some possible non-specific binding of the inhibitor to sites other than the active center of the enzyme, metabolism of this inhibitor and/or presence of enzyme subtypes was hypothesized, the molecular characteristics of SSAO in these tissues (Km, Vmax values, enzyme efficiencies, approximate enzyme concentrations, and molecular turnover numbers) towards the substrate kynuramine (0.1 mM) at pH 7.4 and 37 degrees C have been estimated.     

Soluble semicarbazide-sensitive amine oxidase (SSAO) activity is related to oxidative stress and subchronic inflammation in streptozotocin-induced diabetic rats

Diabetes is known to increase the risk of Alzheimer's disease (AD) and vascular dementia via oxidative stress and inflammation. There are speculations that SSAO activity might be related to the development of AD. Our aim was to investigate whether changes of soluble SSAO activity, oxidative stress and inflammation markers are related to each other in diabetes. Soluble and tissue-bound SSAO activities (from serum and aorta, respectively) were determined in streptozotocin (STZ)-induced diabetic rats without insulin treatment, receiving insulin once, or twice daily compared to control animals. After three weeks of treatment soluble and tissue-bound SSAO activities (seSSAO and aoSSAO, respectively), serum total antioxidant status (TAS), high sensitivity C-reactive protein (hsCRP), fructose amine levels and routine laboratory parameters were determined. SeSSAO activity significantly increased in the diabetic groups without treatment and receiving insulin once daily, and a marked decrease in aoSSAO activity was seen in all diabetic groups. Increased oxidative stress was correlated with hsCRP elevation, while hsCRP and seSSAO activity were also significantly correlated. In all groups seSSAO and aoSSAO activities were in negative correlation with each other. Our results support the view that poor metabolic control leads to increased oxidative stress, which in turn may cause the elevation of hsCRP levels. Soluble SSAO on the one hand acts as an adhesion molecule - thus possibly being a factor responsible for the late complications of diabetes - and on the other hand, it may contribute to oxidative stress. Our parsimonious conclusion is that there is a relation between the risk factors of AD and vascular dementia (diabetes, oxidative stress and chronic inflammation) and SSAO activity, which may originate from the vessel wall.     

Inhibition of murine N1-acetylated polyamine oxidase by an acetylenic amine and the allenic amine, MDL 72527

Murine N¹-acetylated polyamine oxidase (mPAO) was treated with N,N′-bis-(prop-2-ynyl)-1,4-diaminobutane, a poor substrate and inhibitor for the enzyme, with K_m and K_i values in the millimolar range. Apparently, its oxidation produces prop-2-ynal, which reacts with amino acyl nucleophiles. Using a steady-state kinetic assay, four phases were identified, the first being the oxidation of the compound via Michealis-Menten-type kinetics. As prop-2-ynal accumulates, there is a biphasic reduction in the rate. This process leads to an mPAO form that is nearly inactive (fourth phase), but displays classical Michealis-Menten-type kinetics. The enzyme-bound flavin is not modified in this process. In contrast, micromolar concentrations of the MDL 72527 (N,N′-bis-[buta-2,3-dienyl]-1,4-diaminobutane) inhibited mPAO rapidly and completely. It inhibits by first binding tightly and apparently irreversibly, and then slowly converts to a species where the inhibitor is covalently bound to the N5-position of the flavin’s isoalloxazine ring. The covalent adduct was identified as a flavocyanine.     

Hypochlorous acid generates Nε-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Synthetic liver X receptor agonist T0901317 inhibits semicarbazide-sensitive amine oxidase gene expression and activity in apolipoprotein E knockout mice

Semicarbazide-sensitive amine oxidase (SSAO) catalyzes oxidative deamination of primary aromatic and aliphatic amines. Increased SSAO activity has been found in atherosclerosis and diabetes mellitus. We hypothesize that the anti-atherogenic effect of liver X receptors (LXRs) might be related to the inhibition of SSAO gene expression and its activity. In this study, we investigated the effect of LXRagonist T0901317 on SSAO gene expression and its activity in apolipoprotein E knockout (apoE^−/−) mice. Male apoE^−/− mice (8 weeks old) were randomly divided into four groups: basal control group; vehicle group; prevention group; and treatment group. SSAO gene expression was analyzed by real-time quantitative polymerase chain reaction and its activity was determined. The activity of superoxide dismutase and content of malondialdehyde in the aorta and liver were also determined. In T0901317-treated mice, SSAO gene expression was significantly decreased in the aorta, liver, small intestine, and brain. SSAO activities in serum and in these tissues were also inhibited. The amount of superoxide dismutase in the aorta and liver of the prevention group and treatment group was significantly higher compared with the vehicle group ( P < 0.05). Malondialdehyde in the tissues of these two groups was significantly lower compared with the vehicle group ( P < 0.05). Our results showed that T0901317 inhibits SSAO gene expression and its activity in atherogenic apoE^−/− mice. The atheroprotective effect of LXR agonist T0901317 is related to the inhibition of SSAO gene expression and its activity.     

Enzymatic synthesis and characterization of N5-(carboxymethyl)-L-ornithine and N6-(carboxymethyl)-L-lysine

Summary This report describes the enzyme-catalyzed synthesis, characterization, and chromatographic separation of N⁶-(carboxymethyl)-L-lysine and N⁵-(carboxymethyl)-L-ornithine. The two N -(carboxyalkyl)amino acids are formed via a reductive condensation between glyoxylate and the- or-amino groups of lysine and ornithine, respectively. Both reactions are catalyzed by the NADPH-dependent enzyme, N⁵-(carboxyethyl)ornithine synthase [EC 1.5.1.24], found in some strains of the lactic acid bacteriumLactococcus lactis subsp.lactis.     

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.     

Mitochondrial cytochrome c oxidase as a target site for cephalosporin antibiotics in renal epithelial cells (LLC-PK(1)) and renal cortex

We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.     

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.     

Control of lysyl oxidase activity through site-specific deuteration of lysine

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d₂-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V_max/K_m as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D₂Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies.     

An autoantibody against N-(carboxyethyl)lysine (CEL): Possible involvement in the removal of CEL-modified proteins by macrophages

Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N^ε-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when ¹²⁵I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of ¹²⁵I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.     

Properties of copper-free pig kidney amine oxidase: role of topa quinone

Copper removal from pig kidney amine oxidase containing Cu/topaquinone (TPQ) has been obtained using CN(-) in the presence of the poor substrate p-(dimethylamino)benzylamine. Upon removal of copper, the enzyme loses its activity while the TPQ cofactor remains in its oxidized form. The addition of copper to the apo-form fully restores the active enzyme. The CN(-) treatment in the presence of sodium dithionite or good substrates (cadaverine or benzylamine) also removes copper but the TPQ cofactor is irreversibly reduced and the addition of copper does not regenerate the active enzyme. Ni(II) and Zn(II) do not bind the apo-protein in contrast to Co(II) which is incorporated to the same extent as Cu(II). However, Co-reconstituted enzyme only shows a very low activity. These results demonstrate that copper is essential for the catalytic mechanism because it maintains the correct active site geometry.     

Probing electron transfer reactions between two azurins from Alcaligenes xylosoxidans GIFU 1051 with optically active Ru complexes as molecular recognition probes: Importance of the 43rd residue

Electron transfer reactions between optically-active Ru^II/III complexes incorporating (S)-/(R)-amino acids, and the two azurins, azurin-1 (az-1Cu) and azurin-2 (az-2Cu) isolated from Alcaligenes xylosoxidans GIFU 1051, have been studied to probe molecular recognition sites on the two azurins. The Ru^II/III complexes are K[Ru^II(L)(bpy)] and [Ru^III(L)(bpy)], and have a tripodal ligand (L) derived from the (S)-/(R)-amino acids, which are in turn exchanged for other functional substituent groups, such as (S)-/(R)-phenylalanine, -leucine, -valine, -alanine, and -glutamic acid (L = (S)-/(R)-BCMPA, -BCMLE, -BCMVA, -BCMAL, and -BCMGA). In the oxidation reaction of az-1Cu^I promoted by the Ru^III complexes, the kinetic parameters exhibited enantio- and stereo-selectivities, while the same reaction of az-2Cu^I was less enantio- and stereo-selective. These differences suggest that the processes of formation of the activated states are different for the two azurins. On the other hand, such a difference has not been observed for az-1 and az-2 with respect to the reduction reactions promoted by both azurins Cu^II by the Ru^II complexes within the experimental error. This suggests that the neutrality of the Ru complexes is important for precise molecular recognition of azurins. His117 has been proposed as the electron transfer site. The local structures in the vicinity of the His117 side chain in the two azurins, are essentially identical with the exception of the 43rd residue, Val43 and Ala43 for az-1 and az-2, respectively. Electron transfer reactions between Ru^III complexes and a mutant azurin, V43A-az-1, were also carried out. Interestingly, the activation parameters estimated were very similar to those of az-2, indicating that the 43rd residue acts as the electron transfer site in azurins and provides rationalization for the different mechanisms of az-1 and az-2 in redox reactions.     

The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Metabolic profiling using combined GC-MS and LC-MS provides a systems understanding of aristolochic acid-induced nephrotoxicity in rat

We present here a combined GC-MS and LC-MS metabolic profiling approach to unraveling the pathological outcomes of aristolochic acid (AA)-induced nephrotoxicity. Urine samples were analyzed by GC-MS and LC-MS in combination with pattern recognition techniques, e.g. principal component analysis (PCA), orthogonal projection to latent structure-discriminant analysis. The work indicates that AA-induced acute renal toxicity as evidenced by histopathological examinations could be characterized by systemic disturbance of metabolic network involving free fatty acids generation, energy and amino acids metabolism, and alteration in the structure of gut microbiota. Therefore, this method is potentially applicable to the toxicological study, providing a comprehensive understanding of systems response to xenobiotic intervention.     

Detection of uronic oxidase activity in ripening peaches

Uronic acid oxidase activity was found in an extract from harvested peaches that was incubated with citrus pectin at pH 8.5. The product of this reaction was identified by GC-MS analysis to be galactaric acid. The reaction was linear at 37 degrees C for up to 20 h, and the pH optimum was 8.5. The activity found in firm peaches one day after harvest did not change as the peaches softened over 5 days to eating softness. The incubation conditions were those suitable for monitoring the activity of pectate lyase, but instead of finding an increase in galacturonosyl residue reducing groups due to generation of pectin-derived oligosaccharides, uronic acid oxidase catalyzed the oxidation of the aldehyde reducing functions to carboxyl groups.