Effect of phosphate ion on the activity of bovine plasma amine oxidase.

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.     

Cord blood amine oxidase activities relate to arousal and motor functioning in human newborns

To examine relationships between amine oxidase activities and behavior prior to significant postnatal experience, umbilical cord blood samples were collected from 28 normal infants, and the Brazelton Neonatal Behavioral Assessment Scale was administered within 72 hours of birth. Infants with lower platelet monoamine oxidase (MAO) and/or lower plasma amine oxidase activity were more highly aroused, more motorically active and more difficult to console than those with higher MAO activity. These behavioral characteristics are remarkably similar to the more elaborate affective and social features which have previously been associated with MAO activity differences in adult humans and rhesus monkeys.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

Monoamine and diamine oxidase activities in uremic rats

The activities of monoamine and diamine oxidases in various organs and tissues and the amine levels in plasma and urine were determined in chronically uremic and pair-fed control rats. Plasma amine levels were elevated in uremic animals while the urinary excretion of amines was decreased. In uremic as compared to control animals, monomaine oxidase activity was decreased in kidney and muscle, increased in heart and plasma and not altered in liver and cerebrum. Diamine oxidase activity in uremic rats was decreased in kidney, increased in plasma and unchanged in liver and muscle. These alterations of amine oxidase activities in renal failure may affect the metabolism of many amines and thus contribute to the pathogenesis of the uremic syndrome.     

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.     

Oxidase activity of ceruloplasmin and concentrations of copper and zinc in serum of cancer patients.

A balance between oxidant carcinogens and endogenous antioxidant defence is of particular relevance to the carcinogenesis. Ceruloplasmin (Cp) carries up to 90% of Cu in plasma and performs ferroxidase, antioxidant and amine oxidase activity. Cu and Zn, as trace elements, have been recognized to play an important role as cofactors of SOD. The study presents the relationship of the Cp oxidase activity and concentrations of Cu and Zn in serum of 62 patients with breast (BCA), lung (LCA), gastrointestinal (GICA) and gynecological (GYNCA) cancer. The Cp oxidase activity was determined in serum with o-dianisidine as a substrate. Cu and Zn concentrations in serum were measured by using atomic absorption spectrometry. The results of the study have shown significant increase in the mean serum Cp oxidase activity and total Cu concentrations in all patient groups compared with the control one. The total mean serum Zn concentration was found to be decreased only in LCA group as compared with the control. The effect of the cancer progress on the Cp oxidase activity and concentrations of Cu and Zn was observed within the group of all cancer patients (ALLCA) and within the GICA group. The only significant difference in Cu concentrations among various stages of the disease was observed in GICA between local and distant one. Significant positive correlation coefficients were caLculated for the Cp activity and Cu concentrations in the control group and all patients groups, also according to the cancer progress. Future research is needed to evaLuate the consequences of the elevation of the serum Cp oxidase activity and concentration of Cp, Cu and Zn for the host antioxidant-oxidant balance.     

Inhibition of lymphocyte growth by spermidine in medium containing fetal bovine serum

The effect of spermidine and fetal bovine serum on DNA, RNA, and protein synthesis in phytohemagglutinin-stimulated human lymphocytes was investigated. At 10(-4) M spermidine, DNA, RNA, and protein synthesis ceased and 70% of the original cell population died within 62 hr. Lower spermidine concentrations had no significant effect on DNA and protein synthesis, but caused an early, unexplained increase in the rate of RNA synthesis. Heating at 56 degrees C for 30 min had no effect on the plasma amine oxidase activity in fetal bovine and horse sera but abolished the activity in human plasma. It is concluded that low amounts of aminoaldehydes and acrolein produced by plasma amine oxidase at spermidine concentrations below 10(-4) M do not noticeably alter lymphocyte metabolism. However, the aminoaldehydes and acrolein produced become abruptly cytotoxic at 10(-4) M spermidine.     

Inhibition of plasma membrane NADH oxidase activity and growth of HeLa cells by natural and synthetic retinoids

Several retinoids, both natural and synthetic, were evaluated for their ability to modulate NADH oxidase activity of plasma membranes of cultured HeLa cells and the growth of HeLa cells in culture. Both NADH oxidase activity and the growth of cells were inhibited by the naturally-occurring retinoids all trans-retinoic acid (tretinoin) and retinol as well as by the synthetic retinoids, trans-acitretin, 13-cis-acitretin, etretinate and arotonoid ethylester (Ro 13-6298). For all retinoids tested, inhibition of NADH oxidase activity and inhibition of growth were correlated closely. With tretinoin, etretinate and arotonoid ethylester, NADH oxidase activity and cell growth were inhibited in parallel in proportion to the logarithm of retinoid concentration over the range of concentrations 10-8 to 10-5 M. Approximately 70% inhibition of both NADH oxidase activity and growth was reached at 10 µM. With retinol, trans-acitretin and 13-cis-acitretin, inhibition of NADH oxidase activity and growth also were correlated but maximum inhibition of both was about 40% at 10 µM. The possibility is suggested that inhibition of the plasma membrane NADH oxidase activity by retinoids may be related to their mechanism of inhibition of growth of HeLa cells in culture. (Mol Cell Biochem 166: 101-109, 1997)     

Culture medium enhances semicarbazide-sensitive amine oxidase activity

Components of fetal calf serum (FCS) are known to contribute to growth and maintenance of cultured cells. Fetal calf serum supplementation of media also may contribute to the cytotoxicity of other substances to cells grown in vitro. Semicarbazide-sensitive amine oxidase (SSAO) enzyme, present in FCS, metabolizes primary amines and contributes to amine cytotoxicity in vascular smooth muscle cells (VSMC). In cell culture experiments, the media used may greatly affect enzymic activities such as SSAO. In these studies, the SSAO activity in FCS, cultured rat aortic VSMC, and rat plasma was determined in the presence and absence of various culture media. Semicarbazide-sensitive amine oxidase activity in FCS (5-20 microl) was significantly enhanced (approximately 1.5- to 2-fold) in the presence of various culture media, with Dulbecco modified Eagle medium (DMEM), causing the greatest enhancement. Dulbecco modified Eagle medium enhanced the SSAO activity of cultured VSMC in two of the four passages but reduced activity in two passages. Activity in rat plasma was reduced by approximately 25% in the presence of DMEM. The concentrations of various media components, such as glucose, sodium pyruvate, pyridoxine.HCl, and L-glutamine, were not correlated with enhancement. This study identifies an important enhancement effect of culture media on the FCS enzyme, SSAO, although the media components responsible for the enhancement are yet to be identified.     

Novel copper amine oxidase activity from rat liver mitochondria matrix

Copper containing amine oxidases (Cu-AO) represent a heterogeneous class of enzymes classified as EC 1.4.3.6. The present study reports preliminary results on the presence of a novel amine oxidase activity in rat liver mitochondria lysates. Such enzymatic activity was found in the soluble mitochondrial fraction, obtained by simple osmotic shock. The mitochondrial amine oxidase was isolated by affinity chromatography on a newly synthesised spermine-Sepharose. SDS-PAGE showed a single band at about 60 kDa. Upon chromatographic purification, the enzymatic activity was very labile. The crude enzyme activity was tested by spectrophotometric measurements, determining hydrogen peroxide production following oxidative deamination of different substrates, such as polyamines (spermine, spermidine, putrescine and cadaverine) and monoamines (dopamine and benzylamine). The activity, observed on polyamines and not on monoamines, was inhibited by semicarbazide and azide, but not by pargyline, clorgyline and l-deprenil. Enzyme specificity was tested on several diamines characterized by different carbon atom chain length in the range 2-6 carbon atoms. The highest activity was found with 1,2-diamino-ethane and the highest affinity with 1,5-diamino-pentane. The above reported results suggest the presence of a novel copper-dependent amine oxidase in liver mitochondria matrix.     

The organic functional group in copper-containing amine oxidases. Resonance Raman spectra are consistent with the presence of topa quinone (6-hydroxydopa quinone) in the active site

Resonance Raman spectroscopy has been used to probe the structure of the organic cofactor in copper-containing amine oxidases from bovine plasma, porcine kidney, pea seedlings, and the bacterium Arthrobacter P1. The enzymes were first derivatized with phenylhydrazine or p-nitrophenylhydrazine; resonance Raman spectra were obtained on the intact derivatized enzymes and on a derivatized active-site peptide isolated from bovine plasma amine oxidase. Spectra of the intact amine oxidase phenylhydrazones are practically identical, consistent with the enzymes examined containing a similar cofactor. Only minor frequency shifts and some intensity variations are detected between the resonance Raman spectra of intact bovine plasma amine oxidase and the isolated peptide. These spectral perturbations are attributable to differences in the micro-environment between the intact, folded protein and the isolated small peptide in aqueous solution. This rules out the possibility that a new structure is formed during the isolation of the derivatized active-site peptide. Importantly, the resonance Raman spectra of the phenylhydrazine and p-nitrophenylhydrazine derivatives of the bovine plasma amine oxidase peptide are identical to the spectra of the corresponding derivatives of topa quinone (6-hydroxydopa quinone). Hence these data provide strong, independent support for the recent identification of topa as the organic functional group in bovine plasma amine oxidase (Janes, S. M., Mu, D., Wemmer, D., Smith, A. J., Kaur, S., Maltby, D., Burlingame, A. L., and Klinman, J.P. (1990) Science 248, 981-987).     

Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.     

Biogenesis and turnover of peroxisomes involved in the concurrent oxidation of methanol and methylamine in Hansenula polymorpha

Growth of Hansenula polymorpha in shake flasks and chemostat cultures in the presence of methanol as the sole source of carbon and methylamine as the sole source of nitrogen was associated with the development of peroxisomes in the cells. The organelles were involved in the concurrent oxidation of these two compounds, since they contained both alcohol oxidase and amine oxidase, which are key enzymes in methanol and methylamine metabolism, respectively. In addition catalase was present. Peroxisomes with a completely crystalline substructure were observed in methanol-limited chemostat-grown cells. Amine oxidase probably formed an integral part of these crystalloids, whereas catalase was present in a freely diffusable form. Transfer of cells, grown in a methanol-limited chemostat in the presence of methylamine into glucose/ammonium sulphate media resulted in the loss of both alcohol oxidase and amine oxidase activity from the cells. This process was associated with degradation of the crystalline peroxisomes. However, when cells were transferred into glucose/methylamine media, amine oxidase activity only declined during 2 h after the transfer and thereafter increased again. This subsequent rise in amine oxidase activity was associated with the development of new peroxisomes in the cells in which degradation of the crystalline peroxisomes, originally present, continued. These newly formed organelles probably originated from peroxisomes which had not been affected by degradation. When in the methanollimited chemostat methylamine was replaced by ammonium sulphate, repression of the synthesis of amine oxidase was observed. However, inactivation of this enzyme or degradation of peroxisomes was not detected. The decrease of amine oxidase activity in the culture was accounted for by dilution of enzyme as a result of growth and washout.     

Control of benzylamine oxidase activity in Kluyveromyces fragilis grown on primary amines as nitrogen source

Abstract After growth on a mixture of ammonium and either methylamine or n -butylamine as nitrogen sources, benzylamine oxidase activity in yeasts from a number of different genera was found to be repressed to a lesser extent by ammonium than was methylamine oxidase. Catalase activity was better repressed by ammonium with methylamine as the nitrogen source than with n -butylamine. During growth of Kluyveromyces fragilis on equimolar mixtures of ammonium and an amine as nitrogen sources, benzylamine oxidase synthesis began during the period of exclusive growth on ammonium, and a period of simultaneous use of both nitrogen sources was observed just before the ammonium was exhausted. Addition of ammonium to cultures growing on n -butylamine as nitrogen source had no immediate repressive effect on benzylamine oxidase or catalase synthesis. However, growth on limiting ammonium in the absence of amines did give rise to low levels of amine oxidase and derepression of catalase activity. It is concluded that benzylamine oxidase in yeasts is induced strongly by amines as well as being less strongly repressed by ammonium than methylamine oxidase.     

Increase in putrescine,amine oxidase,and acrolein in plasma of renal failure patients

Since polyamines have been suggested to be one of the uremic "toxins," the levels of each polyamine, its oxidized product, acrolein, and amine oxidase in plasma of patients with renal failure were investigated. The level of putrescine was increased, whereas the level of spermine was decreased in the plasma of patients with renal failure. The patients also had increased serum amine oxidase activity leading to increased degradation of spermine. Both levels of free and protein-conjugated acrolein were also increased in plasma of patients with renal failure. The accumulated acrolein found as protein conjugates was equivalent to 180 microM, which was 6-fold higher than in plasma of normal subjects. It was found that acrolein is mainly produced by polyamine oxidase in plasma. A cell lysate containing polyamine oxidase was cytotoxic in the presence of spermine. Our results indicate that the level of acrolein is well correlated with the degree of seriousness of chronic renal failure.     

成年太行山猕猴(Macaca mulatta tcheliensis)肩关节变量的研究

本项研究对成年太行山猕猴肩关节变量进行了测量和统计,作了异速生长分析,并对太行山猕猴(M.mulatta tcheliensis)、滇南猕猴(M.mulatta mulatta)、川西猕猴(M.mulatta lasiotis)、湘鄂猕猴(M.mulatta littoralis)肩关节变量进行了比较和聚类分析。与上述3个亚种相比,太行山猕猴较短的肩胛骨、较小的锁肱指数、较大的三角肌止点指数和较小的摆荡指数,表明太行山猕猴肩关节形态结构更适应于地面上的活动,说明其在地面上活动较多,在树上活动较少。     

Anatomical distribution of primary amine oxidase activity in four adipose depots and plasma of severely obese women with or without a dysmetabolic profile

Semicarbazide-sensitive amine oxidase (SSAO), identical to primary amine oxidase or vascular adhesion protein-1, is a membrane enzyme that generates hydrogen peroxide. SSAO is highly expressed at the adipocyte surface, and its plasma levels increase with type 2 diabetes. Since visceral adipose tissue (AT) is more tightly associated with obesity complications than subcutaneous (SC) abdominal fat, we compared SSAO activity in plasma and 4 distinct AT locations in 48 severely obese women (body mass index (BMI), averaging 54 ± 11 kg/m²), with or without a dysmetabolic profile. Higher glucose and triacylglycerol levels vs lower high-density lipoprotein (HDL)-cholesterol characterized dysmetabolic women (DYS; n = 25) from non-dysmetabolic (NDYS; n = 23), age- and weight-matched subjects. SC, mesenteric (ME), omental (OM), and round ligament (RL) fat locations were collected during bariatric surgery. SSAO capacity to oxidize up to 1 mM benzylamine was determined in AT and plasma with radiometric and fluorimetric methods. Plasma SSAO was higher in the DYS group. SSAO activity was higher in fat than in plasma, when expressed as radiolabeled benzaldehyde per milligram of protein. In ATs from DYS women, protein content was 10 % higher, and basal hydrogen peroxide release lower than in NDYS subjects, except for RL location. The SSAO affinity towards benzylamine did not exhibit regional variation and was not altered by a dysmetabolic profile (K _m averaging 184 ± 7 μM; n = 183). Although radiometric and fluorimetric methods gave different estimates of oxidase activity, both indicated that AT SSAO activity did not vary according to anatomical location and/or metabolic status in severely obese women.     

Type B monoamine oxidase activity in human brain malignant tumors

An increase of monoamine oxidase (MAO) activity was observed in Central Nervous System (CNS) malignant tumors, but the isoform responsible was not identify (Marcozzi et al., 1985). In the present work we report additional data in order to ascertain whether the type A or B MAO isoform is increased in some malignant human tumors of CNS. In the homogenated tissues the amine oxidase activity was determined by the chemiluminescent method, using different and specific substrates or inhibitors of MAO A and B and copper-dependent enzymes. 19 samples from 4 different types of tumors and relative peritumoral tissues were analysed. The highest activity of was imputable to type B MAO.     

Monoamine oxidase activity in hepatopancreas of Octopus vulgaris

1. Monoamine oxidase activity has been studied in hepatopancreas of Octopus vulgaris using 5-HT and PEA as substrates.2. Time courses of MAO activity against 5-HT and PEA show that the enzyme has higher affinity for PEA than for 5-HT.3. MAO activity against 5-HT appears more sensitive than MAO activity against PEA, to variations of the temperature (range 17–67°C).4. The inhibition curves obtained with clorgyline and deprenyl indicate that MAO activity is due to a single form of the enzyme, not corresponding to type A and type B MAO.5. Semicarbazide 10⁻⁴ M does not affect the deamination of 5-HT and PEA, demonstrating that a semicarbazide-sensitive amine oxidase is not involved in this process.     

Amine oxidase activity in commercial preparations of bovine serum albumin

Amine oxidase activity has been identified in commercial samples of bovine serum albumin (BSA). Benzylamine, phenylethylamines and to a lesser extent, indoleamines, were found to be substrates. The amine oxidase activity was inhibited by semicarbazide and was virtually absent in electrophoretically purified samples. Kinetic analysis of benzylamine deamination and experiments utilizing mixed substrates indicate that more than one catalytic activity may be involved. The results show that amine deamination should be considered as a potential source of error in experiments employing high concentrations of commercially available BSA preparations. This would be of particular importance for $\text{in vitro}$ studies with dopamine since this amine was found to be deaminated at a rapid rate.