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M Da Prada

Publications and source records attributed to M Da Prada.

At least 19 recordsLinked to original sources
PubMed1998

Structure-function relationships of mitochondrial monoamine oxidase A and B: chimaeric enzymes and site-directed mutagenesis studies.

To gain insight into the structure of monoamine oxidases (MAO) A and B, we investigated the properties of various chimaeric enzymes, engineered by moving progressively the junction between the NH2- and the COOH-termini of each MAO form. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimaeras with increasing length of the NH2-terminus of MAO-A (up to position 256) showed a marked decrease in affinity towards substrates and inhibitors. Two sequences, spanning position 62 to 103 and 146 to 220, appeared of particular importance in putatively constituting the binding site of MAO-B. Conversely, the catalytic properties and specificity of MAO-A were insensitive to substitution of both the NH2-(up to position 112) and COOH-termini (from residue 395), but further modification of the central sequence of MAO-A was not compatible with activity. None of the engineered chimaeras showed a shift in substrate and inhibitor specificity. Investigation on MAO-B by site-directed mutagenesis revealed that His382 and Thr158 may represent residues relevant for MAO-B catalytic mechanism.

Amino Acid Substitution↗
PubMed1996

Investigation on the structure of the active site of monoamine oxidase-B by affinity labeling with the selective inhibitor lazabemide and by site-directed mutagenesis.

The structural features of the active site of human monoamine oxidase B (MAO-B) were investigated by affinity labeling and site-directed mutagenesis. The pseudosubstrate inhibitor N-[2-aminoethyl]-5-chloro-2-pyridine carboxamide HCl (lazabemide) can be irreversibly linked to MAO-B by reduction of the enzyme-inhibitor complex with NaBH(3)CN. Analysis of the flavin spectrum of [(3)H]lazabemide-labeled human MAO-B indicated that insertion of the inhibitor did not occur into the isoalloxazine ring of FAD. After trypsin digestion and HPLC peptide mapping of the radiolabeled enzyme, two labeled peptides were observed. Sequence analysis showed that both peptides started at Val371 of human MAO-B. These results indicate that [(3)H]lazabemide is incorporated into the MAO-B peptide stretch containing the FAD-modified Cys397. The function of putative active-site residues contained in this region was investigated by site-directed mutagenesis and expression of the mutant proteins in HEK-293 cells. Substitution of His382 of MAO-B with an Arg greatly reduced the enzymic activity, suggesting that this residue may represent a nucleophile relevant for the MAO-B catalytic mechanism. Whereas it has been shown that mutation of Cys389 with a Ser residue does not markedly affect the activity of the enzyme [Wu, H.-F., Chen, K. and Shih, J.C. (1993) Mol. Pharmacol. 43, 888-893] the mutant carrying an Ala at this position was virtually inactive. Conversely, substitution of Lys386 (to Met) and Ser394 (to Ala) did not markedly modify the kinetic properties of the enzyme. We also report that mutation of MAO-B Thr158 (to Ala) resulted in a dramatic loss of enzymic activity.

Affinity Labels↗
PubMed1996

Molecular neuroanatomy of human monoamine oxidases A and B revealed by quantitative enzyme radioautography and in situ hybridization histochemistry.

Monoamine oxidases are key enzymes in the metabolism of amine neurotransmitters and neuromodulators and are targets for drug therapy in depression, Parkinson's and Alzheimer's diseases. Knowledge of their distribution in the brain is essential to understand their physiological role. To study the regional distribution and abundance of monoamine oxidases A and B in human brain, pituitary and superior cervical ganglion, we used quantitative enzyme radioautography with radioligands [3H]Ro41-1049 and [3H]lazabemide, respectively. Furthermore, 35S-labelled oligonucleotides complementary to isoenzyme messengerRNAs were used to map the cellular location of the respective transcripts in adjacent sections by in situ hybridization histochemistry. A markedly different pattern of distribution of the isoenzymes was observed. Highest levels of monoamine oxidase A were measured in the superior cervical ganglion, locus coeruleus, interpeduncular nucleus and ventromedial hypothalamic nucleus. The corresponding messengerRNA was detected only in the noradrenergic neurons of the superior cervical ganglion and locus coeruleus. In contrast to rat brain, monoamine oxidase B was much more abundant in most human brain regions investigated. Highest levels were measured in the ependyma of ventricles, stria terminalis and in individual hypothalamic neurons. Monoamine oxidase B transcripts were detected in serotoninergic raphe neurons, histaminergic hypothalamic neurons and in dentate gyrus granule cells of the hippocampal formation. We conclude that [3H]Ro41-1049 and [3H]azabemide are extremely useful radioligands for high-resolution analyses of the abundance and distribution of catalytic sites of monoamine oxidases A and B, respectively, in human brain sections. From levels of messenger RNA detected, the cellular sites of synthesis of the isoenzymes are the noradrenergic neurons of the locus coeruleus (for monoamine oxidase A) and the serotoninergic and histaminergic neurons of the raphe and posterior hypothalamus, respectively (for monoamine oxidase B). The combination of quantitative enzyme radioautography with in situ hybridization histochemistry is a useful approach to study, with high resolution, both the physiology and pathophysiology of monoamine oxidases in human brain.

Aged↗
PubMed1995

Cellular expression of mRNAs encoding monoamine oxidases A and B in the rat central nervous system.

Monoamine oxidases A and B (MAO-A and MAO-B) oxidatively deaminate neurotransmitter and xenobiotic amines. The cellular localization of these isoenzymes in the central nervous system (CNS) differs markedly and only partly reflects the distribution of their presumed natural substrates. In the present study, by using in situ hybridization with 35S-labelled oligonucleotide probes, we examined the distribution of mRNAs encoding MAO-A and MAO-B in the rat CNS. Probes for tyrosine hydroxylase, histidine decarboxylase, and tryptophan hydroxylase mRNAs were used to demonstrate the catecholaminergic, histaminergic, or serotoninergic nature of some cell populations in adjacent sections. The radioligands [3H]-Ro 41-1049 and [3H]lazabemide (reversible and selective inhibitors of MAO-A and MAO-B, respectively) were used to reveal the protein distribution by enzyme radioautography. The distribution and abundance of transcripts for both isoenzymes in the tissues investigated differed markedly but, in general, correlated with the protein distribution. MAO-A mRNA and protein were most abundant in noradrenergic neurons. However, moderate levels of transcript expression and protein were also detected in the serotoninergic neurons, and low but significant levels were detected in the dopaminergic neurons. An unexpectedly remarkable degree of hybridization signal was apparent in nonaminergic cell populations, e.g., in the cerebral cortices, the hippocampal formation (CA1-3, dentate gyrus), the cerebellar granule cell layer, and the spinal cord motoneurons. In contrast, MAO-B mRNA and protein were most abundant in serotoninergic and histaminergic neurons, Bergmann glial cells, and circumventricular organs, including the ependyma. MAO-B transcripts were also weakly expressed in nonaminergic cells, e.g., in the hippocampal formation (CA1-2). A further nonneuronal localization of MAO-B transcripts was also resolved, e.g., in the glia limitans, the olfactory nerve layer, and the cerebellar peduncle. These findings reveal further the potential of various cell populations to synthesize the isoenzymes, and homologous (aminergic) and heterologous (nonaminergic) patterns of expression as well as coexpression of MAO mRNAs are described.

Animals↗
PubMed1995

Structure/function relationships of mitochondrial monoamine oxidase A and B chimeric forms.

Monoamine oxidases (MAO) A and B show a high degree of amino acid similarity. Apart from the NH2-terminus, which contains an ADP-binding consensus sequence, little is known about their structural features or the sequences involved in the binding of substrates. In the present paper, we have studied the structure/function relationships of MAOs by constructing 18 different chimeric forms of MAO, engineered by moving progressively the junction between the NH2-terminus of one MAO form with the COOH-terminus of its isoenzyme. After transient expression in HEK-293 cells, the properties of these chimeric enzymes were investigated using both selective and nonselective substrates and inhibitors. Whereas exchange of the ADP-binding sequence did not modify the catalytic properties of either MAO isoforms, chimeras with increasing length of the NH2-terminus of MAO-A (up to residue 256) showed a marked decrease in affinity towards the MAO-B substrate phenylethylamine and the inhibitor N-(2-aminoethyl)-5-chloro-2-pyridine carboxamide . HCl (lazabemide) when compared to wild-type MAO-B. No major changes were observed in the kcat values of these chimeras. From the data obtained, two sequences, i.e. 62-103 and 146-220, appeared of particular importance in constituting the binding site of MAO-B. On the other hand, the catalytic properties and specificity of MAO-A appeared to be relatively insensitive to substitution of both the NH2- (up to position 112) and COOH-termini (from residue 395) of MAO-A with the corresponding MAO-B sequences. However, further modification of the central 283-residue sequence of MAO-A did not appear compatible with enzymic activity. None of the engineered chimeras showed a shift in specificity from one isoform to the other.

Base Sequence↗
PubMed1995

Effects of tolcapone, a novel catechol-O-methyltransferase inhibitor, on striatal metabolism of L-dopa and dopamine in rats.

In vivo brain microdialysis was used to assess the effects of tolcapone, a novel central and peripheral inhibitor of catechol-O-methyltransferase on striatal 3,4-dihydroxyphenyl-L-alanine (L-dopa) and dopamine metabolism. The oral administration of 30 mg/kg of tolcapone failed to change dopamine output but elicited a marked and long-lasting decrease of the extracellular levels of homovanillic acid (HVA) and 3-methoxytyramine with a concomitant increase of 3,4-dihydroxyphenylacetic acid (DOPAC). The administration of L-dopa (20 and 60 mg/kg p.o.) + benserazide (15 mg/kg p.o.) resulted in dose-dependent increase of dialysate levels of L-dopa and 3-O-methyl-DOPA. Tolcapone (30 mg/kg p.o.), given as adjunct to both doses of L-dopa, markedly enhanced the elevation or extracellular L-dopa, while it completely prevented the formation of 3-O-methyl-DOPA. In another experiment, the administration of L-dopa + benserazide (30 + 15 mg/kg p.o.) resulted in increased extracellular levels of dopamine, DOPAC, HVA and 3-methoxytyramine. The co-administration of tolcapone (30 mg/kg p.o.) further increased dopamine and DOPAC levels, whereas HVA and 3-methoxytyramine effluxes were reduced. These findings support the notion that tolcapone has the ability to enhance striatal dopamine neurotransmission by increasing L-dopa bioavailability through peripheral and central inhibition of L-dopa O-methylation, as well as by blocking the central conversion of dopamine into 3-methoxytyramine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1995

Integrated pharmacokinetics and pharmacodynamics of the novel catechol-O-methyltransferase inhibitor tolcapone during first administration to humans.

OBJECTIVES: To assess the tolerability, pharmacokinetics and pharmacodynamics of single oral doses of the novel catechol-O-methyltransferase (COMT) inhibitor tolcapone in healthy volunteers. METHODS: In this double-blind, placebo-controlled, ascending-single-dose study, doses of 5 to 800 mg tolcapone were administered orally to eight sequential groups of six young healthy male volunteers. Adverse events, vital signs, and clinical laboratory variables were recorded. Pharmacokinetic parameters of tolcapone and its 3-O-methylmetabolite were determined. Pharmacodynamics were assessed by determination of COMT activity in erythrocytes. RESULTS: Tolcapone was well tolerated at all dose levels and did not exert a detectable influence on vital sign measurements. The drug was rapidly absorbed and showed dose-proportional pharmacokinetics. Its mean elimination half-life was 2.0 +/- 0.8 hours (n = 42). Plasma levels of the 3-O-methylmetabolite of tolcapone were not proportional to dose, and its formation was delayed at higher doses. Its elimination half-life was 32 +/- 7 hours (n = 29). Tolcapone caused a rapid and reversible inhibition of COMT activity in erythrocytes. At doses of 200 mg and higher, COMT activity was inhibited by more than 80%. The pharmacokinetic-pharmacodynamic relationship could be described by an inhibitory Emax model and suggested that metabolites of tolcapone did not substantially contribute to its inhibitory activity. CONCLUSIONS: The novel COMT inhibitor tolcapone was well tolerated at oral doses of 5 to 800 mg. Tolcapone concentration-dependently inhibited COMT activity in erythrocytes and exhibited dose-proportional kinetics. Further investigations into its applicability in the treatment of Parkinson's disease are warranted.

Administration, Oral↗
PubMed1995

Pharmacokinetic-pharmacodynamic interaction between the COMT inhibitor tolcapone and single-dose levodopa.

1. Single oral doses of the catechol-O-methyltransferase (COMT) inhibitor tolcapone (10-800 mg) or placebo were administered simultaneously with a dose of levodopa/benserazide 100/25 mg to seven sequential groups of six healthy male subjects in a two-way crossover study. 2. Plasma concentrations of tolcapone, its metabolite 3-O-methyltolcapone, levodopa and 3-O-methyldopa (3-OMD) were determined in conjunction with COMT activity in erythrocytes. 3. The drug combination was well tolerated at all dose levels and there were no signs indicative of an increase in dopaminergic stimulation. 4. Tolcapone caused a rapid and reversible inhibition of COMT activity in erythrocytes in parallel with a dose-dependent decrease in the formation of 3-OMD. Tolcapone increased the area under the concentration-time curve and elimination half-life of levodopa. The maximum effects were obtained at a dose of about 200 mg when both parameters increased approximately twofold. The drug had no influence on the maximum concentration of levodopa. 5. Tolcapone was rapidly absorbed and eliminated with, on average, a tmax of 1.5 h and a t1/2 of 2.3 h. The drug showed dose-proportional pharmacokinetics, in contrast to 3-O-methyltolcapone whose formation was relatively decreased at higher doses. 6. Plasma concentrations of tolcapone correlated with inhibition of COMT activity in erythrocytes and suppression of 3-OMD levels, but not with changes in levodopa pharmacokinetics.

Adult↗
PubMed1995

The role of monoamine oxidase and catechol O-methyltransferase in dopaminergic neurotransmission.

The action of dopamine (DA) released in the synaptic cleft is mainly terminated by its reuptake and catabolism by the enzymes monoamine oxidase (MAO) and catechol O-methyltransferase (COMT). Preclinical data show that the reduction of the catabolism of DA elicited by MAO and COMT inhibitors leads to an enhancement of DA neurotransmission. Moreover, there is evidence suggesting that MAO-B inhibition might protect DA neurons from oxidative stress. Nevertheless, due to differences in enzyme localization and activity between man and rodents, results obtained in experimental animals might not reflect the actual situation in humans. Today the availability of potent and selective MAO and COMT inhibitors makes it feasible for the clinician to test whether the blockade of catabolic enzymes would result in a symptomatic improvement in Parkinsonian patients, and whether MAO-B inhibition might additionally exert a neuroprotective effect.

Catechol O-Methyltransferase↗
PubMed1994

Radioautographic evidence that the GABAA receptor antagonist SR 95531 is a substrate inhibitor of MAO-A in the rat and human locus coeruleus.

The locus coeruleus (LC), a major noradrenergic nucleus in the brain, probably has a functional role in the regulation of anxiety states as well as vigilance, attention, learning and memory. LC neurons are under the inhibitory control of gamma-aminobutyric acid (GABA) via ionotropic GABAA receptors. However, to date, little is known of the receptor binding characteristics of these neurons. In the present investigation we therefore examined by receptor radioautography the localization of the binding sites for different components of the GABAA receptor complex in the rat and human LC. Both rat and human LC neurons have a high density of binding sites for the pyridazinyl-GABA derivative [3H]SR 95531 (gabazine, a GABAA receptor antagonist for low affinity GABA recognition sites). However, at the concentrations used, no binding sites in the LC were detectable for the benzodiazepine receptor antagonist [3H]flumazenil, the GABAA receptor agonist (for high affinity sites) [3H]muscimol or the ionophore ligand [35S]t-butyl bicyclophosphorothionate (TBPS). Unexpectedly, the pharmacological specificity of [3H]SR 95531 binding to the LC differed markedly from that to most brain regions (IC50 values for GABA and RU 5135 respectively in the LC were > 10(-2) and 10(-3) M; and, for example, in the dentate gyrus the most labelled structure after the LC, 8 x 10(-7) and 1.8 x 10(-9) M). These differences prompted the further characterization of [3H]SR 95531 binding in the LC, revealing a significant affinity for monoamine oxidase type A (MAO-A), which is highly concentrated in this nucleus. In a competition binding study, a reduction of up to 25% of the [3H]SR 95531 binding was observed with MAO-A but not MAO-B inhibitors, at concentrations which produce maximum but selective enzyme inhibition. Correspondingly, 2 h after the oral administration of supramaximal doses of the MAO-A inhibitors moclobemide and Ro 41-1049 (but not the MAO-B inhibitor lazabemide) the in vitro binding of [3H]SR 95531 was markedly reduced (by 77 and 82% of controls respectively). Moreover, enzyme radioautography with [3H]Ro 41-1049 revealed that SR 95531 has a significant affinity for MAO-A (IC50 values were 10(-5) and 4 x 10(-6) M in the LC and dentate gyrus respectively) but not for MAO-B ([3H]lazabemide binding). Altogether, these findings suggest that the high-affinity binding of [3H]SR 95531 to the LC mainly reflects its affinity for MAO-A, which questions its utility as a selective ligand for low-affinity GABA recognition sites in the CNS.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Molecular cloning and functional expression of human 3-hydroxyanthranilic-acid dioxygenase.

Increased cerebral levels of the endogenous excitotoxin quinolinic acid (QUIN) have been speculatively linked to neuronal damage following neurological and inflammatory disorders. 3-Hydroxyanthranilic-acid dioxygenase (3-HAO; 3-hydroxyanthranilate 3,4-dioxygenase, EC 1.13.11.6) is the enzyme that catalyzes the synthesis of QUIN from 3-hydroxyanthranilic acid, and evidence suggests that it could play a role in disorders associated with altered tissue levels of QUIN. In this report, we describe the isolation of a full-length cDNA clone encoding human 3-HAO (h3-HAO). Degenerate oligonucleotides were designed from the amino acid sequences of tryptic peptides of rat liver 3-HAO, and they were used as primers for reverse transcription-polymerase chain reaction of rat liver RNA. The resulting rat cDNA product was used to screen a human hepatoma cell line (HepG2) cDNA library and to isolate a human 3-HAO cDNA clone. This clone was found to have an insert of 1276 nucleotides. The deduced primary structure of h3-HAO is composed of 286 amino acid residues with a predicted molecular mass of approximately 32.6 kDa. The human sequence exhibits high similarity (94%) to the rat partial amino acid sequence deduced from the rat reverse transcription-polymerase chain reaction fragment. Insertion of the h3-HAO coding sequence into a eukaryotic expression vector yielded relatively high amounts of the active enzyme in human embryonic kidney HEK-293 cells. The Km value of 3-HANA for recombinant h3-HAO (approximately 2 microM) was in good agreement with that reported for the native enzyme. Immunoblot analysis of recombinant h3-HAO revealed a polypeptide with an apparent molecular mass of 32 kDa, as predicted from the deduced amino acid sequence. RNA blot analysis of human liver and HepG2 cells revealed one major species of h3-HAO mRNA of approximately 1.3 kilobases.

3-Hydroxyanthranilate 3,4-Dioxygenase↗
PubMed1994

Species differences in changes of heart monoamine oxidase activities with age.

It has previously been established that MAO-A activity markedly increases (about 600%) with age in the rat heart. The aim of the present study was to examine the effect of age on the activity of MAO-A and -B in the mouse heart. In contrast to the rat heart, beta-phenylethylamine is deaminated by MAO-B in the mouse heart. Heart MAO-B activity was found to significantly increase (about 70%) with age in both male and female mice, whereas MAO-A activity remained unchanged. Compared to age-matched rats, serotonin-deaminating activity was about 35 times and 204 times lower in young and old mice, respectively.

Aging↗
PubMed1994

Lazabemide (Ro 19-6327), a reversible and highly sensitive MAO-B inhibitor: preclinical and clinical findings.

Ro 19-6327 (lazabemide, L), MDL 72974, selegiline, AGN 1135 and MDL 72145 were investigated for their MAO inhibitory effect in rat tissues in vitro. The selectivity of MAO-B inhibition of L, selegiline and MDL 72974 was also measured in vitro in human brain tissue as well as ex vivo in rat brain and liver after acute and subchronic administration. Of all compounds investigated L was the most selective for MAO-B inhibition under in vitro and ex vivo conditions. In volunteers, L completely but reversibly inhibited platelet MAO-B with a dose-dependent duration. Clinical trials with L are under way in both Alzheimer's and Parkinson's disease (PD).

Animals↗
PubMed1994

Increased monoamine oxidase B activity in plaque-associated astrocytes of Alzheimer brains revealed by quantitative enzyme radioautography.

The aetiology and pathogenesis of Alzheimer's disease are currently poorly understood, but symptomatic disease is associated with amyloid plaques, neurofibrillary tangles, neuronal loss and numerous alterations of neurotransmitter systems in the CNS. Monoamine oxidase type B is known to be increased in Alzheimer diseased brains. The distribution and abundance of catalytic sites for monoamine oxidases A and B in post mortem human brains of 11 Alzheimer disease cases and five age-matched controls were investigated by quantitative enzyme radioautography. Using tritiated monoamine oxidase inhibitors (Ro41-1049 and lazabemide)--as high affinity substrates selective for monoamine oxidases A and B, respectively--it was found that monoamine oxidase B activity increased up to three-fold exclusively in temporal, parietal and frontal cortices of Alzheimer disease cases compared with controls. This increase was restricted to discrete patches (approximately 185 microns in diameter) which occupied approximately 12% of the cortical areas examined. In other brain regions (hippocampal formation >> caudate-putamen > cerebellum), patches of [3H]lazabemide-enriched binding were less abundant. [3H]Ro41-1049 binding (i.e. monoamine oxidase A) was unchanged in all tissues of diseased versus control brains. The monoamine oxidase B-enriched patches in all cortical regions correlated, in their distribution and frequency, with glial fibrillary acidic protein-immunoreactive clusters of astrocytes. Diffuse and mature beta-amyloid-immunoreactive senile plaques as well as patches of high density binding of [3H]PK-11195--a high-affinity ligand for peripheral-type (mitochondrial) benzodiazepine binding sites in microglia/macrophages--were found throughout Alzheimer diseased cortices. The up-regulation of monoamine oxidase B in plaque-associated astrocytes in Alzheimer's disease--in analogy to its proposed role in neurodegenerative disorders such as Parkinson's disease--might, indirectly, be a potential source of cytotoxic free radicals. Lazabemide, a selective reversible monoamine oxidase B inhibitor, is currently under clinical evaluation for the treatment of Parkinson's and Alzheimer's diseases. We conclude that enzyme radioautography with [3H]lazabemide is a reliable high resolution assay for plaque-associated astroglioses in Alzheimer's disease. Its clinical diagnostic utility for positron emission tomography or single photon emission computer tomography studies is being investigated.

Aged↗
PubMed1994

Estimation of rate constants for serotonin uptake and compartmentation in normal human platelets.

Biochemical and pharmacological experiments led to a model describing a thrombin-releasable (vesicular) compartment and two non-thrombin-releasable compartments for 5-hydroxytryptamine (5-HT, serotonin) in normal human platelets (J. L. Costa, K. L. Kirk, D. L. Murphy, and H. Stark. Br. J. Pharmacol. 72: 449-459, 1981). With tritiated 5-HT as substrate, the amounts of total and nonreleasable intraplatelet tritiated material were measured. Moreover, in the last set of experiments, the amounts of extraplatelet tritiated material and extraplatelet tritiated 5-HT were also measured. With these data, identification of the kinetic constants for movement between the various compartments was possible. A recent extension of the similarity transformation approach to nonlinear compartmental models was used to carry out the a priori identifiability analysis. Then numerical identification was performed by implementing and achieving efficient methods from optimal control theory. Under our experimental conditions, the main results are as follows: 1) the vesicular compartment corresponds to the platelet storage 5-HT compartment, 2) instead of pools I and II proposed previously, only a single nonreleasable thrombin-resistant pool could be detected, and 3) the optimized model computed to suit the first set of experiments fits homologous intraplatelet results obtained under similar experimental conditions in the last set of experiments and also fits one of the two supplementary extraplatelet investigations.

Blood Platelets↗
PubMed1993

Characterisation of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line.

Monoamine oxidase (MAO)-A and MAO-B are FAD-containing mitochondrial enzymes which catabolize biogenic and xenobiotic amines. The N-terminal regions of both forms of MAO contain an ADP-binding consensus sequence found in several dinucleotide-dependent enzymes, but otherwise show remarkable sequence differences. In order to investigate whether the N-terminal region of MAOs participates in the different catalytic properties and inhibitor specificities exhibited by MAO-A and MAO-B, we constructed chimeric A/B forms and expressed them in a human embryonic kidney cell line (293 cells). The MAO-A chimeric form containing the N-terminus (36 amino acids) of MAO-B and the B chimera having the first 45 amino acid sequence of MAO-A were both catalytically active. Compared to the respective wild-type form, they did not show any significant difference in their catalytic properties (Km, kcat) towards the substrates tested or in their sensitivity towards inhibitors. This indicates that the N-terminal region of the two isoenzymes is not involved in the different specificities of MAO-A and MAO-B. Substitution of Cys-397 of MAO-B, i.e. the residue covalently anchoring FAD, with an Ala or a His residue resulted in the total loss of enzymatic activity, suggesting that the covalent coupling of FAD to MAO occurs specifically at the-SH group of cysteine.

Cells, Cultured↗