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.     

Partial amino acid sequence analysis of human placenta monoamine oxidase A and bovine liver monoamine oxidase B

We have prepared peptide maps from human placenta monoamine oxidase type A (MAO-A) and bovine monoamine oxidase type B (MAO-B) and determined the amino acid sequences of 21 of these peptides. These sequences have been compared to the cDNA deduced amino acid sequences of human MAO-A and -B. A result of special interest is the identification of two sets of MAO-A peptides which have sequences different from those deduced from cDNA sequences. This observation is consistent with the notion that MAO-A may be composed of at two subunits which are similar but not identical in primary amino acid sequence.     

Studies on the mechanism of MPTP oxidation by human liver monoamine oxidase B

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its deuterated analogues were oxidized to their corresponding dihydropyridinium species (MPDP+) by preparations of pure human liver MAO B:monoclonal antibody complex to investigate the mechanism of MPTP activation. Lineweaver-Burk plots of initial reaction rates revealed that the Km,app values for the various deuterated MPTP analogues were similar to those of MPTP. In contrast, Vmax,app values were substantially decreased by substitution of deuterium for hydrogen on the tetrahydropyridinium ring, especially at C-6. Deuterium substitution on the N-methyl group alone did not significantly reduce Vmax,app. These studies support the interpretation that oxidation of MPTP at the C-6 position on the tetrahydropyridine ring is a major rate-determining step in its biotransformation by MAO B.     

Effects of tricyclic antidepressants upon human platelet monoamine oxidase

Tricyclic antidepressant drugs were found to inhibit human platelet MAO. The I50 for the inhibition by amitriptyline was 4 × 10^?6 M, 1.6 × 10^?5 M, and 2 × 10^?4 M when phenylethylamine, tryptamine, and benzylamine were used as substrates. Amitriptyline exhibited noncompetitive inhibition with the substrates phenylethylamine and tryptamine but competitive inhibition with benzylamine. Solubilization and partial purification of platelet MAO did not alter the inhibitory effects of tricyclics. Treatment of the partially purified enzyme with the chaotropic agent sodium perchlorate produced only a slight increase in the inhibition constant for amitriptyline. Our findings suggest that selective inhibition of phenylethylamine oxidation may mediate the antidepressant actions of tricyclics. In addition, our studies provide some evidence for the existence of multiple catalytic sites of MAO activity in the human platelet.     

Effect of neurocatin on the activity of monoamine oxidase B in rat brain synaptosomes

Neurocatin, a small (about 2,000 Dalton) neuroregulator isolated from mammalian brain, is a powerful effector of monoamine oxidase B in rat brain synaptosomes. Incubation of intact synaptosomes with neurocatin caused an inhibition of the enzyme dependent on the concentration of neurocatin. This inhibition became statistically significant at a neurocatin concentration of 10 ng/200 l and was significant at all higher neurocatin concentrations. At 40 ng/200 l, neurocatin inhibited monoamine oxidase B activity by about 60%. This inhibitory effect was almost completely abolished by breaking the synaptosomal membrane by hypotonic buffer prior to incubation with neurocatin. In addition, incubation of the synaptosomes in calcium free medium almost completely abolished the inhibitory effect of neurocatin on monoamine oxidase B. The inhibition appeared to involve covalent modification of the enzyme mediated by a neurocatin receptor(s). Measurements of the kinetic parameters of the enzyme showed that 20 ng of neurocatin caused a statistically significant decrease in V_max (by 20%) with no significant change in K_M, compared to controls. Inhibition of monoamine oxidase by neurocatin is potentially of great clinical importance because this enzyme plays a major role in catabolism of the biogenic amines and alterations in its activity is believed to contribute to several neurological disorders.     

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.     

Measurement of platelet monoamine oxidase activity in healthy human volunteers

Platelet MAO activity of 176 healthy volunteers was studied. Activity of the enzyme was found intraindividually stable, repeated measurements in the same individuals performed 3 weeks and one year later revealed only minimum changes. Platelet MAO activity was found to be significantly higher in females than in males. No age dependence of the measured values could be demonstrated. Enzyme activity values determined with different substrates showed a significant positive correlation. A significant activation of the enzyme was found after the addition of platelet poor plasma to the platelet preparation.     

Influence of culture conditions on monoamine oxidase A and B activity in rat astrocytes

Astroglial cells dispersed from newborn rat hemispheres were established in medium supplemented with 20 per cent fetal calf serum (FBS) and then grown to a confluent monolayer in the presence of 10 per cent FBS or charcoal-stripped FBS (CS). Type 1 astrocytes were subcultured and either maintained under the same conditions of the primary cultures or converted to serum-free chemically defined medium (CDM). No differences were found in either MAO A or MAO B activity of astrocytes grown in the presence of FBS or CS after 15 and 21 days in vitro (day 1 and 6 of subculture). In contrast, on day 21 both MAO A and MAO B activities were markedly higher in astrocytes subcultured in CDM compared with cells maintained in serum-supplemented medium. This difference appeared to be due to increased number of enzyme molecules, since kinetic analysis showed an increase in V_max of both MAO isoenzymes in serum-free medium, but no change in K_m. Consistently, the recovery of MAO A and MAO B activity after irreversible enzyme inhibition by clorgyline and deprenyl was faster in CDM than in FBS-supplemented medium, indicating enhanced enzyme synthesis under serum-free condition. Estimates of half-lives for the recovery of MAO A and MAO B activity indicated that, under both culture conditions, type A activity had a higher turnover rate than type B. The effect of CDM on astrocyte MAO does not appear to be due to selection of a subpopulation of cells, but rather linked to a morphological change (differentiation) with increased synthesis of both MAO isoenzymes.     

Increased human platelet monoamine oxidase activity during lithium carbonate therapy

Human platelet monoamine oxidase (MAO) activity was assessed, using benzylamine C¹⁴ as the substrate, in ten patients who received a trial of lithium carbonate treatment. In all ten patients there was an increase of the median MAO activity during the lithium treatment period in comparison to the median MAO activity during a placebo treatment period. This increase in platelet MAO activity is consistent with reports by other investigators that lithium treatment is associated with increased oxidative deamination of monoamines in both animals and humans.     

Iron modulates the activity of monoamine oxidase B in SH-SY5Y cells

Both monoamine oxidase B (MAO-B) and iron accumulation are associated with neurologic diseases including Parkinson’s disease. However, the association of iron with MAO-B activity was poorly understood. Here we took advantage of highly sensitive and specific fluorescence probes to examine the change in MAO-B activity in human dopaminergic neuroblastoma (SH-SY5Y) cells upon iron exposure. Both ferric and ferrous ions could significantly enhance the activity of MAO-B, instead of MAO-A, in SH-SY5Y cells. In addition, iron-induced increase in MAO-B probe fluorescence could be prevented by pargyline and other newly developed MAO-B inhibitors, suggesting that it was MAO-B activity-dependent. These findings may suggest MAO-B is an important sensor in iron-stressed neuronal cells.     

High-level expression of human liver monoamine oxidase B in Pichia pastoris

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.     

Chromenylchalcones with inhibitory effects on monoamine oxidase B

Structure–activity relationship (SAR) calculations were used to find monoamine oxidase-B (MAO-B) inhibitors by identifying pharmacophores exhibiting high inhibitory activities. Several such chromenylchalcones were designed and synthesized accordingly. Their inhibitory effects on MAO-B were determined using an HPLC-based method and an MAO-B enzyme assay kit. (E)-3-(6-Methoxy-2H-chromen-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one exhibited a half-maximal inhibitory concentration of 320 nM. Its molecular-level binding mode with the three-dimensional structure of MAO-B was elucidated using an in silico docking study. The chromenylchalcone scaffold, which is derived from natural products including isoflavonoids and chalcones, had not been previously reported as an MAO-B inhibitor.     

An enzyme immunoassay for the measurement of human monoamine oxidase B protein.

A heterologous, competitive, solid phase ELISA has been developed which can measure monoamine oxidase (MAO) B concentration (both inactive and active) in human platelets and other tissue extracts. The assay is based on competition between a soluble form of MAO and MAO bound to a solid phase for binding to a limiting amount of a MAO B-specific monoclonal antibody, 3F12/G10. It utilises a crude and easily prepared sample of human liver mitochondrial membranes as the source of solid phase MAO. Optimal assay conditions allow detection of MAO B down to at least 0.5 ng (4.18 fmol; 6.25 ng/ml) of protein. As the assay is sensitive and simple to operate, it will allow a systematic assessment of the role of platelet MAO concentrations in the aetiology of psychiatric and neurologic conditions.     

Effects of amiridin and tacrine, drugs effective in Alzheimer's disease, on the activity of monoamine oxidase A and B]

In vitro comparative studies of effects of amiridin (9-amino-2, 3, 5, 6, 7, 8-hexahydro-1H-cyclopentane (b) choline monohydrate hydrochloride) and tacrine physostigmine and piracetam on monoamine oxidase A (MAO-A) and B (MAO-B) activity in the rat brain were carried out. Piracetam (1 x 10(-4)-1 x 10(-3) M) dose-dependently increased MAO-A and MAO-B activity. At all concentrations used (1 x 10(-7)-5 x 10(-4) M) physostigmine had no effect on MAO-A and MAO-B activity. Amiridin was found to inhibit MAO-B activity at 5 x 10(-4) M concentration only. Tacrine inhibited MAO-A activity at 5 x 10(-4) M concentration. The therapeutical effects of amiridin and tacrine in treatment of Alzheimer disease were not related to their action on MAO-A and -B activity.     

Effects of L-DOPA administration upon monoamine oxidase activity in rat tissues

The effects of L-DOPA administration in various doses (250,500 and 1000 mg/kg for 7 days) upon type A and B MAO activities in rat tissues have been investigated using the substrates 5-HT, tyramine and benzylamine. The specific activities of MAO in heart, kidney and brain were significantly increased after L-DOPA, whereas liver and vas deferens MAO was unchanged. None of the observed changes was totally specific for either form of the enzyme, although some evidence for a slight selectivity on type A MAO of heart and type B MAO of kidney and brain was obtained. These results indicate that some tissues may respond to elevated intracellular catecholamine levels, resulting from L-DOPA administration, by increasing their capacity to deaminate those amines. However, at present, the exact mechanism by which these changes are brought about is unknown.     

Modification of substrate-inhibitor affinities of human platelet monoamine oxidase B in vitro

The rate of benzylamine utilization by monoamine oxidase (MAO)-B from human blood platelets was 2-4 times higher than that for octopamine. Both activities were inhibited 100% by 10(-7) M deprenyl (a specific MAO-B inhibitor) and were not affected by clorgyline (a specific MAO-A inhibitor) or by polyclonal antibodies to MAO-A. The preincubation of platelet MAO-B with purified MAO-A from mitochondrial membranes of human placenta resulted in appearance of excess octopamine activity. This additional activity was not precipitated by antibodies to MAO-A or inhibited by deprenyl but was inhibited by clorgyline. Incubation of the MAO-A preparation from placenta at 45 degrees C for 15 min before its preincubation with MAO-B caused 50% loss of both activities. Protease inhibitors had no effect on the modification of MAO. These data indicate that MAO-A or a factor tightly bound to it can modify MAO-B yielding a form of the enzyme with both MAO-A and MAO-B substrate and inhibitor affinities and MAO-B immunospecificity.