The decrease of NAD(P)H has a prominent role in dopamine toxicity.

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.

Characterization of monoamine oxidase activity present in human granulocytes and lymphocytes.

The characterization of monoamine oxidase (MAO) activity in lymphocytes and granulocytes was studied by using cells prepared from human blood. The specific activities of the enzyme towards beta-phenylethylamine (PEA), benzylamine (Bz), tyramine (TYR) and 5-hydroxytryptamine (5-HT) were found to be 5-times higher in lymphocytes than in granulocytes. The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by the lack of effect of semicarbazide on the benzylamine oxidation. The presence of MAO-B was corroborated by the inhibition of PEA oxidation with nanomolar deprenyl concentrations and by inhibition of TYR oxidation with high clorgyline concentrations, as well as by the simple sigmoid curve obtained in both cases. These results, together with the substrate preferences, suggest that the MAO activity of human granulocytes and lymphocytes is predominantly of the B form. For each fraction the kinetic constants were determined towards PEA, TYR and Bz as substrates. The Km values were similar for both cellular samples, whereas the Vmax values were higher in lymphocytes than in granulocytes. MAO-B was titrated with [3H]pargyline in order to find out the number of active sites. The corresponding molecular concentration, Kcat values and turnover number showed the presence of related enzymes in human granulocytes and lymphocytes.

Protamines and other basic proteins from spermatozoa of molluscs.

The basic proteins obtained from spermatozoa of different species of the phylum Mollusca have been extracted and fractionated. The amino acid analysis and electrophoretic mobility of these proteins show a considerable variation in the types and relative amounts of the components present in different species. In some case-(Gibbula, Haliotis, Loligo, Octopus) the main components are similar to the protamines found in the salmonid fishes, although they appear to be larger in size (40-80 amino acids) and show significant differences in amino acid composition. In other cases (Mytilus, Chiton) a complex mixture of proteins is present, which including somatic-like histones and proteins intermediate in size and composition betweeln protamines and histones. Other molluscs (Ostrea, Spisula, Patella) also contain proteins intermediate in composition between protamines and histones, but their molecular weight appears to be larger than in histones. In Eledone a complex mixture of proteins containing cystine is obtained, with some components rich in arginine. In most species, somatic-like histones are also present. Their type and relative amount are different in each species. The significance of these results towards an understanding of the evolutionary history of these proteins is discussed. It is suggested that these proteins evolved from histone precursors.

Activation of human lung semicarbazide sensitive amine oxidase by a low molecular weight component present in human plasma.

Semicarbazide-sensitive amine oxidase (SSAO) encodes a wide family of enzymes named E.C.1.4.3.6 [amine:oxygen oxidoreductase (deaminating) (copper containing)] that metabolises primary aliphatic and aromatic amines. It is present in almost all vascularised and nonvascularised mammalian tissues, and it is also present in soluble form in plasma. SSAO appears to show different functions depending on the tissue where it is expressed. Here we describe, for the first time, the activation of the SSAO from human lung by human plasma. The extent of activation was greater when the human plasma came from diabetic and heart infarcted patients. A kinetic mechanism of such effect is proposed. The activation was lost after the plasma was dialysed, indicating a low molecular weight component (MW <3800 Da) to be responsible. The activator component is heat stable and resistant to proteolysis by chymotrypsin and trypsin and also resistant to perchloric acid treatment. However, treatment with 35% formic acid, completely abolished activation, suggesting involvement of lipid material. The possibility of that lysophosphatidylcholine (LPC), an amphiphilic phospholipid derived from the phosphatidylcholine, the major component in plasma accumulated in pathological conditions, was studied. LPC was shown to behave as a "competitive activator" of human lung SSAO at concentrations below its critical micellar concentration (CMC value=50 microM). Thus LPC may be a component of the SSAO activatory material present in human plasma.

Protective effect of the multitarget compound DPH-4 on human SSAO/VAP-1-expressing hCMEC/D3 cells under oxygen-glucose deprivation conditions: an in vitro experimental model of cerebral ischaemia.

BACKGROUND AND PURPOSE: Stroke and Alzheimer's disease (AD) are related pathologies in which the cerebrovascular system is involved. Plasma levels of semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1, also known as Primary Amine Oxidase -PrAO) are increased in both stroke and AD patients and contribute to the vascular damage. During inflammation, its enzymatic activity mediates leukocyte recruitment to the injured tissue, inducing damage in the blood-brain barrier (BBB) and neuronal tissue. We hypothesized that by altering cerebrovascular function, SSAO/VAP-1 might play a role in the stroke-AD transition. Therefore, we evaluated the protective effect of the novel multitarget-directed ligand DPH-4, initially designed for AD therapy, on the BBB. EXPERIMENTAL APPROACH: A human microvascular brain endothelial cell line expressing human SSAO/VAP-1 was generated, as the expression of SSAO/VAP-1 is lost in cultured cells. To simulate ischaemic damage, these cells were subjected to oxygen and glucose deprivation (OGD) and re-oxygenation conditions. The protective role of DPH-4 was then evaluated in the presence of methylamine, an SSAO substrate, and/or ß-amyloid (Aß). KEY RESULTS: Under our conditions, DPH-4 protected brain endothelial cells from OGD and re-oxygenation-induced damage, and also decreased SSAO-dependent leukocyte adhesion. DPH-4 was also effective at preventing the damage induced by OGD and re-oxygenation in the presence of Aß as a model of AD pathology. CONCLUSIONS AND IMPLICATIONS: From these results, we concluded that the multitarget compound DPH-4 might be of therapeutic benefit to delay the onset and/or progression of the neurological pathologies associated with stroke and AD, which appear to be linked.

Molecular determinants of MAO selectivity in a series of indolylmethylamine derivatives: biological activities, 3D-QSAR/CoMFA analysis, and computational simulation of ligand recognition.

A series of indolylmethylamine derivatives were assayed toward MAO-A and MAO-B inhibition. The K(i) values of these compounds are in the range from 0.8 to >10(6) nM for MAO-A or from 0.75 to 476000 nM for MAO-B. The most selective MAO-A or MAO-B inhibitors elicit a ratio of K(i) in the order of 1500 or 1000, respectively. Comparison of MAO-A and MAO-B CoMFA models showed that both the steric and electrostatic properties at the 5 position of the indole ring are determinant for MAO selectivity. Computational simulations of the complex between this part of the ligand and Phe-208 of MAO-A or Ile-199 of MAO-B, experimentally identified as responsible for substrate selectivity, allowed us to further characterize the nature of these enzyme-inhibitor interactions.

Tissue activity and cellular localization of human semicarbazide-sensitive amine oxidase.

Semicarbazide-sensitive amine oxidase (SSAO), widely distributed in highly vascularized mammalian tissues, metabolizes endogenous and xenobiotic aromatic and aliphatic monoamines. To assess whether its physiological role in humans is restricted to oxidation, we used an immunohistochemical approach to examine the cellular localization of SSAO in human peripheral tissues (adrenal gland, duodenum, heart, kidney, lung, liver, pancreas, spleen, thyroid gland, and blood vessels) and also analyzed its subcellular localization. The results are in agreement with the specific activities also determined in the same samples and are discussed with reference to the tissue distribution of monoamine oxidase A and B. Together with the oxidative deamination of monoamines, SSAO cellular localization indicates that, in most human peripheral tissues, it might participate in the regulation of physiological processes via H(2)O(2) generation. (J Histochem Cytochem 49:209-217, 2001)

Relevance of benzyloxy group in 2-indolyl methylamines in the selective MAO-B inhibition.

1. Previous studies with indolyl derivatives as monoamine oxidase (MAO) inhibitors have shown the relevance of the indole structure for recognition by the active site of this enzyme. We now report a new series of molecules with structural features which determine the selectivity of MAO inhibition. 2. A benzyloxy group attached at position 5 of the indole ring is critical for this selective behaviour. Amongst all of these benzyloxy-indolyl methylamines, N-(2-propynyl)-2-(5-benzyloxyindol)methylamine FA-73 was the most potent MAO-B 'suicide' inhibitor studied. 3. The Ki values for MAO-A and MAO-B were 800+/-60 and 0.75+/-0.15 nM, respectively. These data represent a selectivity value of 1066 for MAO-B, being 48 times more selective than L-deprenyl (Ki values of 376+/-0.032 and 16.8+/-0.1 nM for MAO A and MAO-B, respectively). The IC50 values for dopamine uptake in striatal synaptosomal fractions from rats were 150+/-8 microM for FA-73 and 68 +/- 10 microM for L-deprenyl whereas in human caudate tissue the IC50 values were 0.36+/-0.015 microM for FA-73 and 0.10+/-0.007 microM for L-deprenyl. Moreover, mouse brain MAO-B activity was 90% ex vivo inhibited by both compounds 1 h after 4 mg kg(-1) administration, MAO-A activity was not affected. 4. These novel molecules should provide a better understanding of the active site of monoamine oxidase and could be the starting point for the design of further selective, non-amphetamine-like MAO-B inhibitors with therapeutic potential for the treatment of neurological disorders.

Inhibition of monoamine oxidase A and B activities by imidazol(ine)/guanidine drugs, nature of the interaction and distinction from I2-imidazoline receptors in rat liver.

1. I2-Imidazoline sites ([3H]-idazoxan binding) have been identified on monoamine oxidase (MAO) and proposed to modulate the activity of the enzyme through an allosteric inhibitory mechanism (Tesson et al., 1995). The main aim of this study was to assess the inhibitory effects and nature of the inhibition of imidazol(ine)/guanidine drugs on rat liver MAO-A and MAO-B isoforms and to compare their inhibitory potencies with their affinities for the sites labelled by [3H]-clonidine in the same tissue. 2. Competition for [3H]-clonidine binding in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the pharmacological profile of the interaction (2-styryl-2-imidazoline, LSL 61112 > idazoxan > 2-benzofuranyl-2-imidazoline, 2-BFI = cirazoline > guanabenz > oxymetazoline > > clonidine) was typical of that for I2-sites. 3. Clonidine inhibited rat liver MAO-A and MAO-B activities with very low potency (IC50S: 700 microM and 6 mM, respectively) and displayed the typical pattern of competitive enzyme inhibition (lineweaver-Burk plots: increased K(m) and unchanged Vmax values). Other imidazol(ine)/guanidine drugs also were weak MAO inhibitors with the exception of guanabenz, 2-BFI and cirazoline on MAO-A (IC50S: 4-11 microM) and 2-benzofuranyl-2-imidazol (LSL 60101) on MAO-B (IC50: 16 microM). Idazoxan was a full inhibitor although with rather low potency, on both MAO-A and MAO-B isoenzymes (IC50S: 280 microM and 624 microM, respectively). Kinetic analyses of MAO-A inhibition by these drugs revealed that the interactions were competitive. For the same drugs acting on MAO-B the interactions were of the mixed type inhibition (increased K(m) and decreased Vmax values), although the greater inhibitory effects on the apparent value of Vmax/K(m) than on the Vmax value indicated that the competitive element of the MAO-B inhibition predominated. 4. Competition for [3H]-Ro 41-1049 binding to MAO-A or [3H]-Ro 19-6327 binding to MAO-B in rat liver mitochondrial fractions by imidazol(ine)/guanidine compounds revealed that the drug inhibition constants (Ki values) were similar to the IC50 values displayed for the inhibition of MAO-A or MAO-B activities In fact, very good correlations were obtained when the affinities of drugs at MAO-A or MAO-B catalytic sites were correlated with their potencies in inhibiting MAO-A (r = 0.92) or MAO-B (r = 0.99) activity. This further suggested a direct drug interaction with the catalytic sites of MAO-A and MAO-B isoforms. 5. No significant correlations were found when the potencies of imidazol(ine)/guanidine drugs at the high affinity site (pKiH, nanomolar range) or the low-affinity site (pKiL, micromolar range) of I2-imidazoline receptors labelled with [3H]-clonidine were correlated with the pIC50 values of the same drugs for inhibition of MAO-A or MAO-B activity. These discrepancies indicated that I2-imidazoline receptors are not directly related to the site of action of these drugs on MAO activity in rat liver mitochondrial fractions. 6. Although these studies cannot exclude the presence of additional binding sites on MAO that do not affect the activity of the enzyme, they would suggest that I2-imidazoline receptors represent molecular species that are distinct from MAO.

Monoamine oxidase activities in lymphocytes and granulocytes taken from pig blood.

The characterisation of monoamine oxidase activities in lymphocytes and granulocytes was studied using cells prepared from pig blood. The specific activities against beta-phenylethylamine, benzylamine, tyramine and 5-hydroxytryptamine as substrates in granulocytes (G) were approximately twice those found in lymphocytes (L). The absence of the semicarbazide-sensitive amine oxidase (SSAO) was confirmed by insensitivity of the latter to semicarbazide as inhibitor with benzylamine as substrate. MAO activity present in (G) and (L) was selectively inhibited by low deprenyl concentrations; this fact, in addition to the simple sigmoid inhibition curves obtained with increasing concentrations of clorgyline with tyramine as substrate, suggests that the MAO activity present both in (G) and (L) is predominantly of the MAO-B form. The absence of any contamination with plasma amine oxidase (EC 1.4.3.6) was confirmed by the fact that activity towards benzylamine (Bz) was insensitive to KCN-induced inhibition. Kinetic constants were determined for each fraction towards beta-phenylethylamine (PEA) and Bz as substrates. MAO-B was titrated with unlabelled pargyline, deprenyl and [3H]-pargyline; the corresponding Kcat values, turnover number and the active concentrations were then determined. The molecular weight of MAO-B present in both cellular fractions was calculated by SDS-electrophoresis and fluorography, after reaction with [3H]-pargyline. Some of these results are compared with those obtained with human blood leucocytes.

A comparative study of some kinetic and molecular properties of microsomal and mitochondrial monoamine oxidase.

This experimental work tries to characterize the monoamine oxidase of microsomal origin through its kinetic and molecular properties, and to establish a comparative study with the enzyme present in rat liver mitochondria. The temperature effect upon this catalytic activity was examined and similar behaviour of MAO A and MAO B between both cellular fractions was found. The study of the pH dependence of initial velocity showed similar results both in mitochondria and in microsomes. The FAD cofactor is covalently attached to the MAO of microsomal origin. The FAD containing subunits corresponding to MAO A and MAO B, previous binding of the enzyme with [3H]pargyline and posterior SDS electrophoresis and fluorography, showed molecular weights of 65,900 and 62,400, respectively, in both cellular fractions. The inhibition curves with clorgyline, deprenyl, semicarbazide and KCN, measuring the remaining activity towards 1 microM of benzylamine, indicated that in mitochondria 5% of the total activity is due to the presence of SSAO activity whereas in microsomes this activity represents about 20%. From all these results it appears that mitochondrial and microsomal MAO are related enzymes, although further structural studies are necessary to confirm their possible identity.

Effects of nondenaturating zwitterionic detergent CHAPS, 3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulfonate, on rat liver mitochondrial and microsomal monoamine oxidase.

It has been reported that monoamine oxidase (MAO) activity (EC1.4.3.4) and, in general, enzymes possessing cationic substrates, were activated and inhibited by anionic and cationic detergents, respectively. In order to examine this hypothesis, the effect of the zwitterionic detergent CHAPS 3-[(3-cholamido propyl) dimethyl ammonio]-1-propanesulphonate was studied in comparison with the effects of cationic, anionic, and non-ionic detergents. The non-denaturating zwitterionic detergent CHAPS was used to solubilise rat liver monoamine oxidase MAO (EC1.4.3.4) of mitochondrial and microsomal origin; the solubilisation conditions, purification, inhibition and kinetic studies were then determined. These results are compared with those previously obtained with the non-ionic detergent Triton X-100, which would also be expected to have no net charge, and are interpreted in terms of specific ionic effects.

The oxidation of dopamine by the semicarbazide-sensitive amine oxidase (SSAO) from rat vas deferens.

The activities of monoamine oxidase A and B and the semicarbazide-sensitive amine oxidase from rat vas deferens were compared towards benzylamine and dopamine. The selective inhibitors (-)-deprenyl and clorgyline were used to allow the contributions of the A and B forms of monoamine oxidase to be determined separately. Comparison of the kinetic constants of the three enzymes towards dopamine indicated that, although each of them had activity towards this substrate, their relative contributions to the total oxidative deamination would depend on the substrate concentration. At all concentrations in the range 1 microM to 10 mM monoamine oxidase-B would contribute about 50% of the total activity. In the range 1 to 10 microM the contributions made by activities of monoamine oxidase-A and the semicarbazide-sensitive enzyme were similar but at higher concentrations the activity of the latter enzyme became more important, its contribution to the total activity rising to some 35% of the total at 500 microM dopamine. The activity of the semicarbazide-sensitive enzyme towards dopamine might thus be important under conditions where either or both the monoamine oxidases were inhibited in pharmacological studies. Its possible relevance to Norrie disease, in which both forms of the human enzyme are deficient, requires further examination.

Contribution of different amine oxidases to the metabolism of dopamine in bovine retina.

The contribution of monoamine oxidase (MAO) A, MAO B and semicarbazide-sensitive amine oxidase (SSAO) to the metabolism of dopamine in the bovine retina was studied. These activities were present in the optic nerve, iris, choroid and bovine retina, but they were absent in the lens. SSAO activity towards dopamine was present in the choroid and the retina, but not in the iris or the optic nerve. The corresponding kinetic values for this substrate in the retina and the choroid showed higher affinity for MAO A (Km 271 and 197 microM, respectively) than for MAO B (Km 861 and 404 microM, respectively). This effect was counteracted by the higher Vmax value for MAO B resulting in the Vmax/Km ratio being similar for both cases. The absence of detectable SSAO activity towards dopamine in these last two tissues contrasts with the presence of that enzyme when benzylamine was studied as substrate. These results indicate that two different SSAO activities could be present in the bovine eye.

Amine oxidase activities in rat breast cancer induced experimentally with 7,12-dimethylbenz(alpha)anthracene.

The activities and distribution of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) in solid breast tumour induced in the rat by treatment with 7,12-dimethylbenz(alpha)anthracene (DMBA) were studied. The mammary tumours were classified according to anatomopathological criteria into: the benign fibroadenoma (FAD) and the malignant adenocarcinoma (ADC) and infiltrant adenocarcinoma (I-ADC). The proportions of total MAO (15%) and SSAO activities (85%) did not change with malignancy. However, an increasing degree of malignancy was associated with an increase in MAO-A activity and a decrease in MAO-B and SSAO activities. Kinetic constants were calculated for SSAO and for each MAO form separately, using specific substrates. The Km values did not change significantly with the degree of malignancy, but Vmax values for MAO-A increased whereas Vmax for SSAO and MAO-B diminished with malignancy. The dependence of SSAO activity on protein concentration indicated the presence of endogenous reversible inhibitory material in extracts from the more malign tumours. This inhibitor was associated with the microsomal fraction and was not removed by dialysis. It was also present in detergent-solubilized extracts, suggesting that the phenomenon might be due to an association of the enzyme itself producing an inactive species.

Evidence of a coupled mechanism between monoamine oxidase and peroxidase in the metabolism of tyramine by rat intestinal mitochondria.

The relationship between monoamine oxidase (EC 1.4.3.4; MAO) and peroxidase (EC 1.11.1.7; POD) in the metabolism of tyramine was investigated using the crude mitochondrial fraction of rat intestine. When tyramine was incubated with mitochondria, the formation of the peroxidase-catalysed oxidation product, 2,2'-dihydroxy-5,5'-bis(ethylamino)diphenyl (dityramine), identified by mass spectrometric analysis, was monitored spectrophotometrically. After an initial lag time, the formation rate of dityramine was linear up to 2 hr, amounting to 17 nmol x hr(-1) x mg protein(-1). A similar value was found for the oxidative deamination of tyramine catalysed by intestinal MAO. Either 10(-3) M clorgyline or 10(-3) M NaCN suppressed this reaction by completely inhibiting MAO or POD, respectively. In the former case, however, addition of H2O2 to the incubation mixture promptly started the reaction. Selective inhibition of MAO-A and MAO-B was achieved with 3 x 10(-7) M clorgyline and 3 x 10(-7) M deprenyl, respectively, and the formation rate of dityramine decreased in a corresponding manner. Preincubation with histamine or spermidine reduced the lag time without affecting the steady-state reaction rate. Higher levels of dityramine were also detected in vivo in rat intestine after oral administration of tyramine. These results indicate that the peroxidase-dependent metabolism of tyramine in the gut may be driven by H2O2 produced by MAO activities and that MAO-A is mainly responsible for this process, as well as for the oxidative deamination of tyramine.

The effect of side chain substitution at positions 2 and 3 of the heterocyclic ring of N-acetylenic analogues of tryptamine as monoamine oxidase inhibitors.

N-Acetylenic analogues of tryptamine in which the side chain is located at position 2 of the indole ring are compared with those in which the side chain is located at position 3, in terms of their actions as inhibitors of monoamine oxidases A and B. IC50 values at 0 and 30 min of pre-incubation were determined. Time-dependence and irreversible inhibition confirmed that all of them behave as mechanism-based inhibitors. The kinetic constants of each inhibition step were determined for both monoamine oxidase forms and compared between them. In all cases the first-order rate constants for the covalent adduct formation were similar to inhibitor selectivity which is derived solely from differences in affinities for non-covalent binding to the A and B enzymes. Those compounds where the acetylenic side chain was substituted at position 2 of the heterocyclic ring and selective inhibitors of monoamine oxidase A were more potent than those with the side chain in position 3.

Determination of monoamine oxidase concentrations in rat liver by inhibitor binding.

The concentrations of monoamine oxidase-A and -B have been determined in mitochondria, mitochondrial outer membranes and microsomes from Sprague-Dawley and Wistar rats by determining the binding of tritium-labelled pargyline. Although the amounts of each form present depended on the source and the preparation method, this was paralleled by the specific activity such that the molecular turnover number was found to remain constant. The catalytic constants, kcat/Km, which represents the apparent second-order rate constant for the combination of enzyme and substrate, were about 0.13 and 2.1 sec-1 X microM-1 for 5-hydroxytryptamine and 2-phenethylamine, respectively, regardless of the source. Estimations of the amounts of the two forms by determining the concentrations of the inhibitors clorgyline, (-)-deprenyl, J-508 or pargyline necessary to give complete inhibition were shown to give overestimates of the true values because of the non-specific binding of these inhibitors to sites other than the monoamine oxidase active site.

Inhibition of bovine lung semicarbazide-sensitive amine oxidase (SSAO) by some hydrazine derivatives.

Microsomal semicarbazide-sensitive amine oxidase (SSAO) from bovine lung was shown to be inhibited by a number of hydrazine derivatives, but the mechanisms of inhibition were found to differ. Hydralazine behaved as an irreversible and partially time-dependent inhibitor with an IC50 value of 1 microM under the conditions used. Phenylhydrazine was found to be a potent irreversible inhibitor of SSAO (IC50 30 nM). Semicarbazide behaved as a specific irreversible inhibitor (active-site-directed irreversible inhibitor) in first forming a non-covalent enzyme-semicarbazide complex (with a Ki value of 85 microM), which then reacted to give an irreversibly inhibited enzyme species in a reaction defined by the first-order rate constant k2 = 0.065 min-1. Phenelzine behaved as a reversible inhibitor, but dialysis at 37 degrees C was found to be necessary to obtain full recovery of enzyme activity. The dependence of inhibition on phenelzine concentration was complex and consistent with multiple binding sites for this inhibitor. This diversity in the action of a family of compounds with the same functional group must be taken into account in attempts to design more specific inhibitors of this enzyme.

Semicarbazide-sensitive amine oxidase (SSAO) from human and bovine cerebrovascular tissues: biochemical and immunohistological characterization.

Semicarbazide-sensitive amine oxidase (SSAO) is widely distributed in almost all tissues, especially in vascularized ones. However, its presence in brain microvessels is still controversial. We have investigated the presence of SSAO in human and bovine brain microvessels by biochemical and immunohistological techniques, and we have compared it with SSAO present in meninges from the same species. SSAO metabolizes benzylamine and methylamine in all tissues tested and possibly dopamine and octopamine as well, as shown in competition studies. Kynuramine inhibited the metabolism of benzylamine by SSAO with high affinity in a non-competitive manner. Western-blot analysis rendered a positive staining of a 100 kDa band, in tissues from both species. These results were confirmed by immunohistological studies: the tunica media and intima of the meninges from both species were positively stained, and so was the endothelial layer of microvessels. SSAO was absent in brain parenchyma. These results definitively confirm the presence of SSAO in human and bovine cerebrovascular tissues and they demonstrate for the first time, the presence of this amine oxidase in endothelial cells from microvessels, through biochemical and immunological approaches.