Binding and deamination of various substrates by types A and B monoamine oxidase in bovine brain mitochondria

The binding and deamination of four substrates by type A and type B monoamine oxidase (MAO) in bovine brain mitochondria were investigated in mixed substrate experiments. MAO activity in bovine brain mitochondria, with 5-hydroxytryptamine (5-HT) as substrate, was highly sensitive to clorgyline and less sensitive to deprenyl, while MAO activity with benzylamine or β-phenylethylamine (PEA) as substrate was highly sensitive to deprenyl and less sensitive to clorgyline. On the other hand, when tyramine plus PEA was used as substrate, the inhibition curves of clorgyline and deprenyl were both biphasic. These results indicate that 5-HT and benzylamine were preferentially deaminated by type A MAO and type B MAO, respectively, and that tyramine and PEA were deaminated by both types of MAO. Studies on the inhibition by clorgyline plus deprenyl of tyramine deamination (in the absence and presence of another substrate) showed that the deamination of tyramine by both type A and type B MAO was inhibited by PEA or benzylamine, while only type A MAO was inhibited significantly by 5-HT. The KA_i value, the dissociation constant of the type A MAO and 5-HT complex, and the KB_i values, the dissociation constants of the type B MAO and PEA or benzylamine complex, were almost equal to the K_m values of type A MAO and type B MAO respectively. The KA_i values for PEA and benzylamine were 78 and 58 μM respectively. For the type B MAO-5-HT complex, the dissociation constant KB_i was 1447 μM. These results show that type A MAO deaminates tyramine and 5-HT whereas benzylamine is not deaminated, but only binds to the substrate binding site of type A MAO with almost the same rate as that for deamination by type B MAO; with type B MAO, tyramine, PEA and benzylamine are deaminated, whereas 5-HT is not deaminated and binds to the substrate binding site of type B MAO with low affinity.     

Possible heterogeneity of type B monoamine oxidase in pig heart mitochondria

The metabolism in vitro of 5-hydroxytryptamine (5-HT), tyramine and benzylamine by pig heart mitochondrial monoamine oxidase (MAO) has been studied. Linear Lineweaver-Burk plots yielded estimated K_m values (at pH 7.8) of 475 μM (5-HT) and 292 μM (tyramine). In contrast, linear regions of a downward-curving reciprocal plot revealed the presence of a high- and low-affinity metabolizing site (estimated K_m of 39 and 853 μm respectively) for benzylamine. Studies with the irreversible MAO inhibitor clorgyline indicated that metabolism of the three substrates in this tissue was brought about by type B MAO alone. However, the apparent sensitivity toward clorgyline of each substrate-metabolizing activity was not identical. This was due to different degrees of rapid or possibly instantaneous inhibition of enzyme activity toward each substrate. This rapid inhibition appeared to be both partially reversible and irreversible to a relative degree depending upon the substrate-metabolizing activity studied; additional time-dependent inhibition developing with prolonged preincubation was a first-order process, with a similar half-life, whichever substrate was used to assay MAO activity. Ackermann-Potter and Lineweaver-Burk plots also demonstrated differences in the inhibitory effects of clorgyline upon metabolism of each substrate. The ability of 5-HT, tyramine and benzylamine to inhibit each other's deamination in vitro was also investigated. Enzyme activity was measured by radiochemical assay with each labeled substrate in the presence and absence of the other non-labeled amines. Lineweaver-Burk analysis revealed a competitive interaction between tyramine and benzylamine, whereas mixed-type inhibition patterns were obtained for mixtures containing 5-HT/tyramine or 5-HT/benzylamine. In this latter case, the present inhibition data could only be assessed accurately with the low-affinity catalytic site for benzylamine. The kinetics of heat denaturation indicated both a thermolabile and thermostable component of each substrate-metabolizing activity. Some substrate-dependent differences in the relative proportions of these components were found. These experiments are discussed in relation to similar studies by other workers and suggest that pig heart MAO may, in fact, be heterogeneous.     

Human brain monoamine oxidase: one molecular entity-multiple binding sites?

Monoamine oxidase (MAO) of human brain cortex was partially characterized by using different substrates and inhibitors. Two Km values were calculated for each of the three substrates tested, i.e., phenethylamine (PEA) benzylamine (BA) and 5-hydroxtryptamine (5-HT). Clorgyline and 5-HT, both known as MAO-A occupants, were able to abolish the second (high) Km deamination of PEA. 5-HT, while non-competitively inhibiting the deamination of low BA concentrations, competitively inhibited the deamination of high concentrations of this type B substrate. The kinetics of 5-HT deamination showed positive cooperation which indicates the involvement of subunits in the enzyme structure. The ability of some phospholipids to change the enzyme behaviour was considered as indication that these molecules might play a role in determining the ratio between the so-called A and B types of MAO, and in the regulation of the enzyme's activity.     

Substrate and inhibitor selectivity of human heart monoamine oxidase.

The subcellular distribution, inhibitor sensitivity, thermostability and pH profiles of monoamine oxidase (MAO) from samples of human heart obtained at post mortem have been investigated with several substrates. A simple subcellular fractionation showed that, with either tyramine or benzylamine as substrate, about 50 per cent of the MAO activity was found in the mitochondrial fraction, with negligible quantities in the high speed supernatant. From the use of clorgyline, it appears that 5-HT is a substrate for MAO-A, benzylamine and β-phenethylamine are substrates for MAO-B, while tyramine and dopamine are substrates for both forms of the enzyme, d-Amphetamine was shown to be a selective competitive inhibitor of MAO-A, of similar potency to that observed with MAO from rat liver. No significant difference between the thermostability at 50° of the MAO activity towards 5-HT and benzylamine was observed. Preliminary results for the effect of pH on human heart MAO are presented. The results are discussed with respect to similar data obtained for MAO from other human and animal tissues.     

酶标仪比色法测定单胺氧化酶活性

目的建立用酶标仪比色测定单胺氧化酶A(monoamine oxidase A,MAO A)和MAO B活性的方法。方法以大鼠肝组织匀浆作酶原,分别以血清素(5-HT)和苄胺作为MAO A和MAO B底物,以2,4-二硝基苯肼(DNPH)和氢氧化钠为显色剂,在450 nm处用酶标仪比色测定。结果该方法能快速、灵敏、稳定、选择性地测定MAO活性。结论建立了以酶标仪比色法测定MAO活性的方法用于筛选药物。     

Human brain monoamine oxidase: one molecular entity-multiple binding sites?

Monoamine oxidase (MAO) of human brain cortex was partially characterized by using different substrates and inhibitors. Two K_m values were calculated for each of the three substrates tested, i.e., phenethylamine (PEA), benzylamine (BA) and 5-hydroxtryptamine (5-HT). Clorgyline and 5-HT, both known as MAO-A occupants, were able to abolish the second (high) K_m deamination of PEA. 5-HT, while non-competitively inhibiting the deamination of low BA concentrations, competitively inhibited the deamination of high concentrations of this type B substrate. The kinetics of 5-HT deamination showed positive cooperation which indicates the involvement of subunits in the enzyme structure. The ability of some phospholipids to change the enzyme behaviour was considered as indication that these molecules might play a role in determining the ratio between the so-called A and B types of MAO, and in the regulation of the enzyme's activity.     

β-Phenylethylamine and benzylamine as substrates for human monoamine oxidase A: A source of some anomalies?

Monoamine oxidase (MAO) A predominates both in human placenta and lung. With 5-hydroxytryptamine (5-HT), β-phenylethylamine (PEA) and benzylamine (Bz) as substrates and clorgyline and deprenyl, respectively, as selective MAO A and B inhibitors, their activity pattern has been defined and compared with that of human liver. PEA had a much higher V_max with placental MAO A than did Bz; it behaved largely as an A substrate in placenta, and partly as an A substrate in lung. At commonly used substrate concentrations, deamination of Bz (sensitive to 10^?7 M deprenyl) was a better indicator of MAO B activity than deamination of PEA. The divergence between PEA and Bz as MAO A and B substrates may be one reason for some of the apparent discrepancies in the behaviour of MAO A and B noted in a variety of tissues in the literature.However, Bz reacts with benzylamine oxidase (BzAO) as well as MAO B. Depending on the tissue, deprenyl-resistant Bz activity may indicate the presence of BzAO rather than MAO A. As there is a widespread distribution of BzAO in man and rat, BzAO should be considered among the alternatives of enzyme activity when Bz is used as substrate.     

Characteristics of monoamine oxidase in mitochondria isolated from chick brain,liver, kidney and heart

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied with mitochondria of chick brain, liver, kidney and heart. The kinetic constants for MAO in these organs were determined, using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For all the substrates, the V_max values were highest in kidney, followed in decreasing order by brain, liver and heart. For tyramine and PEA, the K_m values were lowest in liver, but for 5-HT it was lowest in heart. Inhibition experiments with clorgyline and deprenyl were carried out on mitochondria of the four organs with the three substrates at their K_m concentrations. From the plateaus observed of inhibition by clorgyline, it was concluded that 5-HT was oxidized by both types of MAO in mitochondria of all the organs; PEA was fairly specific for type B MAO in brain, liver and kidney, but non-specific in heart. In heart mitochondria, appreciable amounts of the activities toward tyramine and PEA were due to an amine oxidase distinct from mitochondrial MAO; 5-HT, however, was oxidized exclusively by mitochondrial MAO in this organ. The above atypical characteristics in substrate specificity found in chick tissues support the idea that the type A and type B concept cannot be applied uncritically to all tissues from all species.     

A comparison of the pharmacological and biochemical properties of substrate-selective monoamine oxidase inhibitors

1. M&B 9302, E-250, NSD 2023, and Lilly 51641, substrate-selective inhibitors of monoamine oxidase (MAO), and two non-selective inhibitors of MAO (tranylcypromine and phenelzine) have been compared in the rat for activity in (i) inhibiting rat brain monoamine oxidase in vitro and in vivo using tyramine, 5-hydroxytryptamine (5-HT) and benzylamine as substrates; (ii) increasing brain levels of noradrenaline (NA) and 5-HT and (iii) antagonizing tetrabenazine-induced sedation.2. Concentrations of M&B 9302 and Lilly 51641 required to produce 50% inhibition of 5-HT oxidation by brain mitochondrial MAO were 1.4 x 10(-8)M and 2.5 x 10(-7)M respectively. Higher concentrations were required to inhibit tyramine oxidation whilst benzylamine oxidation was inhibited only at concentrations above 10(-5)M.3. E-250 showed the reverse substrate-selectivity in inhibiting the oxidation of benzylamine at concentrations below that required to inhibit the oxidation of 5-HT. NSD 2023 showed little substrate selectivity in vitro.4. Qualitatively similar results were obtained in vivo, except that NSD 2023 showed more marked substrate-selectivity.5. All the inhibitors except E-250 produced a dose-related rise in brain 5-HT levels. Only phenelzine and Lilly 51641 showed a linear relationship between NA levels and dose.6. All the drugs antagonized, in dose-related fashion, the effects of tetrabenazine in reducing locomotor activity. E-250 and NSD 2023 failed to restore locomotor activity to control levels whilst in high doses the other inhibitors, when given before tetrabenazine, produced a considerable increase in locomotor activity.7. Antagonism of tetrabenazine sedation appears to be correlated with (a) inhibition of the enzyme species that oxidize 5-HT and NA but not with inhibition of the enzyme species that oxidize benzylamine; (b) the rise in brain 5-HT levels rather than NA levels.     

Some interrelated properties of A and B form monoamine oxidase in monkey brain mitochondria

The multiplicity of monoamine oxidase (MAO) in monkey brain was studied by comparing the relationship between the selective substrates of MAO and the pH-activity curves obtained using these substrates. When mitochondrial and A-form MAO were used as the enzyme preparations with serotonin (5-HT) and norepinephrine (NE), preferential substrates for A-form MAO, the pH optima were 8.8 and 7.8 with 5-HT and 8.5 and 7.2 with NE. These substrates were also oxidized by B-form MAO after changing the pH of the incubation medium (shift to alkaline); these pH optima were 9.0 and 8.2, respectively. When common substrates of MAO were used (tyramine, octopamine, dopamine and tryptamine), the pH activity curves obtained were all broad and bell-shaped with pH optima for the 3 species of enzyme (mitochondria, A-form and B-form MAO) at 8.0, 7.8, and 8.0 with tyramine; 8.3, 7.5, and 8.5 with octopamine; 7.8, 7.5, and 8.5 with dopamine; and 8.0, 8.3, and 6.9 with tryptamine, respectively. The pH optima were 6.6 with beta-phenylethylamine (beta-PEA) and 9.0 with benzylamine, preferential substrates for B-form MAO, for either mitochondria or B-form MAO. The Km values obtained for tryptamine and beta-PEA were lower than those for the other substrates of MAO, regardless of the enzyme preparations. The Km and Vmax values of both forms MAO for 5-HT and NE were similar to those of the A-form MAO. The differences in the Km and Vmax values of the A-form MAO and B-form MAO for common substrates were comparable. Tyramine, octopamine and dopamine were substrates for both forms MAO, with only a slight preference for B-form MAO over A-form MAO. However, tryptamine may be deaminated predominantly by A-form MAO.     

Properties of monoamine oxidase in mouse neuroblastoma N1E-115 cells

Monoamine oxidase (MAO) from adrenergic mouse neuroblastoma N1E-115 cells was compared to MAO found in rat and mouse brain, rat superior cervical ganglion, and human platelet. In comparison to MAO from brain and ganglion, mouse neuroblastoma MAO deaminated 5-hydroxytryptamine (5-HT) to a proportionately greater extent than all other substrates studied, with benzylamine deamination representing only 1 per cent that of 5-HT. Neuroblastoma MAO was over 1000 times more sensitive to inhibition by clorgyline than by deprenyl. With increasing concentrations of clorgyline, inhibition of tyramine deamination was represented by a simple sigmoid curve, suggesting the presence of primarily one form of MAO. Our results are consistent with evidence for a specific form of MAO associated with sympathetic neurons and suggest that neutoblastoma N1E-115 cells are highly enriched in MAO type A.     

Properties of a semicarbazide-sensitive amine oxidase in human umbilical artery

The metabolism of some aromatic amines by amine oxidase activities in human umbilical artery homogenates has been studied. The inhibitory effects of clorgyline showed that 5-hydroxytryptamine (5-HT) and tryptamine, 1 mM, were predominantly substrates for monoamine oxidase (MAO) type A, whereas MAO-A and B were both involved in the metabolism of beta-phenylethylamine (PEA), 100 microM, and tyramine, 1 mM. About 20-30% of tyramine and PEA metabolism was resistant to 1 mM clorgyline, but sensitive to inhibition by semicarbazide, 1 mM, indicating the presence of a semicarbazide-sensitive amine oxidase (SSAO). Benzylamine, 1 mM, appeared to be metabolized exclusively by SSAO with a Km (161 microM) at pH 7.8 similar to that found for SSAO in other human tissues. Tyramine and PEA were relatively poor substrates for SSAO, with very high apparent Km values of 17.6 and 13.3 mM, respectively, when determined in the presence of clorgyline, 10(-3) M, added to inhibit any metabolism of those amines by MAO activities. However, kinetic studies with benzylamine indicated that clorgyline, 10(-3) M, also appears to inhibit SSAO competitively such that the true Km values for tyramine and PEA may be about 60% of those apparent values given above. No evidence for the metabolism of 5-HT or tryptamine by SSAO was obtained. The aliphatic amine methylamine was recently shown to be a specific substrate for SSAO in umbilical artery homogenates. We have used benzylamine and methylamine as SSAO substrates in histochemical studies to localize SSAO in tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate- and inhibitor-related characteristics of human platelet monoamine oxidase

Human platelet monoamine oxidase (MAO) preferentially deaminated benzylamine and phenylethylamine, two substrates relatively specific for type B MAO, in comparison to 5-hydroxytryptamine, a substrate specific for type A MAO. In studies comparing human platelet and rat brain MAO specific activities, benzylamine and 5-hydroxytryptamine deamination by platelets was approximately 90 and 2 per cent, respectively, that of brain, while platelet deamination of dopamine, tryptamine and tyramine was 20 per cent or less than that of brain. Among sixteen drugs studied, platelet MAO activity was selectively inhibited by low concentrations of the MAO-B inhibitors, deprenyl and pargyline, and was relatively insensitive to the MAO-A inhibitors, clorgyline and Lilly 51641. These observations, in addition to the simple sigmoid inhibition curves obtained with increasing concentrations of either clorgyline or deprenyl, suggest that platelet MAO consists of essentially one distinguishable form of MAO which most closely resembles the MAO type B found in other tissues.     

Studies on beta-phenylethylamine deamination by human placental monoamine oxidase

Kinetical properties of human placental monoamine oxidase (MAO) were investigated in studies on inhibitors and mixed substrates. MAO activity was determined by a radioisotopic assay. Lineweaver-Burk plots were linear at higher and lower concentrations of PEA, whereas at intermediate substrate concentrations, a downward curving plot was obtained. The Km values of the low- and high-affinity sites for PEA deamination were estimated. Studies with mixed substrates showed that 5-HT was a competitive inhibitor and tyramine a mixed-type inhibitor of deamination at high concentrations of PEA, whereas both were non-competitive inhibitors at lower concentrations of PEA. After pre-incubation of human placental mitochondrial preparations with deprenyl, Lineweaver-Burk plots were completely linear, and the Km value was the same as that obtained at low concentrations of PEA in the absence of deprenyl. Tyramine and 5-HT were competitive inhibitors of PEA deamination by deprenyl-treated MAO. From these results it is concluded that there are two kinds of MAO with high- and low-affinity sites for PEA in mitochondria of human placenta, corresponding to type B and A Mao, and that tyramine, 5-HT and PEA share a substrate-binding site on type A Mao, while tyramine and 5-HT bind to a site on type B MAO that is different from the PEA binding site.     

Characterization of eight biogenic indoleamines as substrates for type A and type B monoamine oxidase

Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, 5-hydroxytryptamine (5-HT), 5-hydroxy-N-methyltryptamine, bufotenine, 5-methoxytryptamine, and 5-methoxy-N,N-dimethyltryptamine were characterized as substrates for type A and type B monoamine oxidase (MAO) in rat liver mitochondria. Experiments on sensitivity to clorgyline and to deprenyl, using two substrate concentrations, showed that tryptamine and its N-methylated and N,N-dimethylated derivatives were common substrates for both types of MAO at a substrate concentration of 20.0 μM; at 1000 μM, tryptamine and N-methyltryptamine were common, but N,N-dimethyltryptamine became specific for type B MAO. All the 5-hydroxy- or 5-methoxy-indole derivatives were almost completely specific for type A MAO at a substrate concentration of 20.0 μM; when the concentration was 1000 μM, some of the MAO activity was due to type B MAO for 5-HT, bufotenine and 5-methoxytryptamine. The rat liver mitochondrial enzyme was pretreated with 10^?7M clorgyline and 10^?7M deprenyl to obtain, respectively, the type B-rich and the type A-rich enzyme. These enzyme preparations were subjected to kinetic analyses for the eight amines. From the kinetic analyses, together with data on inhibitor sensitivity, the following phenomena can be described. N-Methylation of tryptamine or of 5-HT did not cause appreciable changes in the specificity of the substrates toward each type of MAO, but it elevated the K_mvalue of type B MAO when the values for tryptamine and N-methyltryptamine were compared. N,N-Dimethylation of tryptamine and 5-HT tended to increase the specificity for type B MAO. All the dimethylated compounds had very low activities with either type A or type B MAO. Either the 5-hydroxy- or the 5-methoxy-group contributed to the specificity of the substrates for type A MAO.     

Characteristics of mitochondrial and synaptosomal monoamine oxidase in monkey brain

Enzymic properties of monoamine oxidase (MAO) from monkey brain were studied. High MAO activity was observed in the mesencephalon and dienecephalon of the brain. Highest activity in every region of the brain was found with tyramine as a substrate. Monkey brain mitochondrial MAO showed a different substrate specificity and different Km and Vmax values than the enzyme from mice, rats, guinea pigs and rabbits. The pH activity curves were all bell-shaped, but the pH optima were remarkably different with the various substrates used. The activities of various substrates at pH 7.2 were compared with those at the pH optimum. At the pH optima, the activity was about 1.2-fold higher with tyramine and dopamine, 2-fold higher with beta-phenylethylamine (beta-PEA) and 3-fold higher with serotonin (5-HT) and benzylamine. These results were almost similar when synaptosomes from monkey brain were used. MAO activities with 5-HT and beta-PEA were strongly inhibited by much lower concentrations of clorgyline and deprenyl, respectively. Plateau-shaped inhibition curves by these inhibitors were obtained with tyramine as the substrate. These results indicate that both the A- and B-form of MAO appear to be uniformly distributed in monkey brain, and the A-form of MAO represents approximately 35% and 50% of the total MAO activity in mitochondria and synaptosomes, respectively.     

Observations on the inhibition of rat liver monoamine oxidase by clorgyline

Two forms of monoamine oxidase (MAO) denned as MAO A and B by others differ in their specificities to substrates and their sensitivities to the irreversible inhibitor clorgyline. From studies using the substrates 5-HT, tyramine and benzylamine, the presence of both MAO forms in rat liver mitochondria has been confirmed and some characteristics of their inhibition by varying concentrations of clorgyline investigated. Although both MAO forms showed time-dependent inhibition, this process occurred, in general, at a qualitatively slower rate for MAO B, despite the fact that this enzyme form requires higher concentrations of clorgyline than MAO A for inhibition of its activity. However, factors such as the concentration of enzyme, the concentration of clorgyline and the enzyme: drug ratio employed in the assay all influence the resultant time-course and the final degree of the inhibition observed. The possible importance of the lipid environment of the outer mitochondrial membrane in generating multiple MAO forms and in regulating the inhibition kinetics of these forms is discussed. The results indicate that the effects of pre-incubation time and the enzyme: drug ratio on inhibition of MAO by clorgyline should be fully recognized when using the drug to indicate multiple forms in animal tissues.     

Characterization of rat skeletal muscle monoamine oxidase.

Optimal conditions for deamination of 5-hydroxytryptamine in rat skeletal muscle were determined. The presence of monoamine oxidase (MAO) A and MAO B isozymes was demonstrated by the use of tyramine (a substrate of both forms), specific substrates (serotonin and benzylamine), and specific inhibitors (clorgyline and deprenyl) of MAO A and B respectively. A 6.5:3.5 ratio of MAO A to B was found using a whole muscle homogenate, while a 7.5:2.5 ratio was found with isolated mitochondria. Thermal inactivation studies demonstrated that skeletal muscle MAO A is more susceptible to heat inactivation than MAO B. The approximate proportion of muscle homogenate MAO which is present in sympathetic nerves was found to be 18 per cent, as determined by treating rats with 6-hydroxydopamine and quantifying the decrease in activity. Significant inhibition of MAO activity was observed after administration in vivo of the MAO inhibitors pargyline, tranylcypromine and harmaline.     

Alterations in monoamine oxidase activity after single and repeated exposure of rats to chlorphentermine

Monoamine oxidase (MAO) activity was determined in rat tissues following in vivo treatments with chlorphentermine (CP). Oxidation of seven amine substrates by liver, lung or brain mitochondrial MAO was investigated at 2 h after a single i.p. injection (60 mg/kg) or after repeated injection (once daily for 3 days, 20 mg/kg, i.p.). Deamination of the type A substrates, norepinephrine, serotonin and octopamine, was decreased significantly in liver, lung and brain after both single and repeated injections. Oxidative deamination of tyramine and dopamine (type A + B substrates) was also lowered in all organs after single and repeated exposure to CP, but to a lesser degree than the type A substrates. However, oxidation of the type B substrates, benzylamine and tryptamine, was unaffected by CP administration in comparison to control. These data indicate that CP is a specific inhibitor of mitochondrial MAO, form A.     

Distribution and inhibition characteristics of human brain monoamine oxidase.

Monoamine oxidase (MAO) activity in 14 regions of 10 normal post-mortem human brains using 5-hydroxytryptamine (5HT), benzylamine, tyramine and dopamine as substrates is presented. Regional distribution with 5HT, benzylamine and tyramine was generally similar with the highest activities observed in the hypothalamus. However, with dopamine as substrate, highest MAO activity occurred in the nucleus accumbens. Although there was relatively greater MAO activity towards 5HT than towards benzylamine in all four cerebral cortical areas studied compared with the caudate, putamen, accumbens and hypothalamus this apparently greater proportion of type A MAO in cortex could not be confirmed with the use of the specific inhibitor clorgyline. In some cases inhibition curves with clorgyline (and correspondingly with deprenyl) were not the expected double sigmoid shape. It is suggested that characterisation of MAO by techniques dependent on the use of specific inhibitors in samples of human brain collected and stored in the usual manner may prove difficult to interpret.