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)     

Deamination of hordenine by monoamine oxidase and its action on vasa deferentia of the rat

The selectivity of the naturally occurring amine, N,N-dimethyltyramine (hordenine) for monoamine oxidase (MAO) and its action upon isolated vasa deferentia of the rat was investigated. Hordenine was deaminated by rat liver MAO with a Michaelis constant of 479 microM and maximum velocity of 128 nmol (mg protein)-1 h-1 compared with 144 microM and 482 nmol (mg protein)-1 h-1 for tyramine. Studies, with selective irreversible inhibitors of MAO, showed that hordenine was a highly selective substrate for MAO-B of liver and that it was not deaminated by the MAO-A of intestinal epithelium. In contrast to tyramine, hordenine did not produce contractions of isolated vasa deferentia. However, 25 microM hordenine potentiated contractile responses of vasa, from control animals, to submaximal doses of noradrenaline and inhibited responses to tyramine. It did not alter responses, to noradrenaline, of vasa denervated by chronic pretreatment of rats with guanethidine. Therefore, it appears that hordenine acted as an inhibitor of noradrenaline uptake, in isolated vasa deferentia. These results indicate that dietary-hordenine is unlikely to be deaminated by intestinal MAO as this is predominantly MAO-A. Consequently, it is likely to be absorbed and could affect the sympathetic nervous system, by virtue of its action as an inhibitor of noradrenaline uptake.     

Vascular smooth muscle cells: a major source of the semicarbazide-sensitive amine oxidase of the rat aorta

Several methods have been used to study the distribution of the semicarbazide-sensitive amine oxidase (SSAO) within the wall of the rat aorta. After separation of the smooth muscle-containing layers of the tunica media from the connective tissue of the tunica adventitia, much higher specific enzyme activity (measured with 1 microM benzylamine) was found in homogenates of the media than of adventitia. Similar results were obtained for MAO-A (with 1 mM 5-HT as substrate). SSAO activity was also considerably higher in homogenates of cells (predominantly smooth muscle) isolated from medial tissue by enzymatic dissociation with collagenase and elastase compared with homogenates of cells (mostly of connective tissue origin) from the adventitia. Histochemical staining resulting from SSAO activity (with benzylamine as substrate) occurred predominantly and intensely over the tunica media in rat aortic sections, although some occasional staining of adventitial sites was also observed. Staining was prevented by the SSAO inhibitors hydroxylamine (1 microM) and semicarbazide (1 mM), but not by the MAO inhibitor, clorgyline (1 mM). These results indicate that SSAO is associated predominantly, although not exclusively, with the smooth muscle cells in the rat aorta. Our findings that beta-aminopropionitrile (BAPN) is a reversible, competitive inhibitor (Ki around 2 X 10(-4)M) of SSAO, in contrast to the irreversible inhibition of the connective tissue lysyl oxidase by BAPN reported by others, provides further evidence that these enzymes are not identical.     

A comparison of cardiac and vascular clorgyline-resistant amine oxidase and monoamine oxidase: Inhibition by amphetamine,mexiletine and other drugs

Clorgyline-resistant amine oxidase (CRAO) and monoamine oxidase (MAO) were studied in homogenates of rat heart and aorta, using benzylamine and tyramine as substrates. In heart, benzylamine at 0.001 mM was deaminated solely by CRAO. With higher concentrations of benzylamine (0.01, 0.1 and 1.OmM), an increasing involvement of MAO-A and MAO-B became apparent in the deamination of benzylamine such that, at 1.0 mM benzylamine, deaminated products resulted equally from MAO-A, MAO-B and CRAO. In aorta, benzylamine was deaminated solely by CRAO irrespective of the concentration used. Tyramine (0.01, 0.1, 1.0 and 5.0 mM) was deaminated entirely by MAO-A in heart, whereas in the aorta both MAO-A and CRAO participated. In aorta the ratio of product formation from MAO-A and CRAO did not vary with changes in the concentration of tyramine, indicating similar K_m values for both enzymatic activities. Further studies with tyramine (0.1 mM) and clorgyline showed biphasic inhibition curves suggestive of two distinct MAO-A components in both heart and aorta. The two components showed different properties in the heart when compared with aorta. When homogenates of hearts were heated at 50° for 1 hr, their sensitivity to inhibition by clorgyline increased, while in homogenates of aorta sensitivity to clorgyline decreased. CRAO was investigated further with benzylamine as substrate. Kinetic studies gave similar K_m values for both heart and aorta (4–6 μM at pH 7.8), and these values were not altered by flushing the assay tubes with oxygen. However, flushing with nitrogen caused uncompetitive inhibition in the heart and noncompetitive inhibition in aorta. These results suggest a difference in the catalytic mechanism between CRAO of heart and aorta. In both heart and aorta, CRAO was inhibited by semicarbazide, (+)-amphetamine, phenelzine and (+)- and (?)-mexiletine, with the (+)-form being more potent. Straight-chain diamine and polyamine compounds failed to inhibit in concentrations up to 10^?4 M. Thus, CRAO is not a typical diamine or polyamine oxidase. The results show differences between heart and aortic CRAO and MAO-A, and the possibility exists for heterogeneity within each of these two distinct forms of amine oxidase. Additionally, drugs known to inhibit MAO-(+)-amphetamine, phenelzine and mexiletine also inhibit CRAO. However, the biological significance of since the physiological role of CRAO is unknown.     

Substrate specificity and inhibitor sensitivity of monoamine oxidase in rat kidney mitochondria.

The deamination of the substrates 5-hydroxytryptamine (5-HT), tyramine, dopamine, β-phenylethylamine and benzylamine by rat kidney mitochondrial monoamine oxidase (MAO) was studied, and kinetic constants are reported for each substrate. By the use of the selective MAO inhibitors, clorgyline and deprenyl, 5-HT and benzylamine were found to be substrates for types A and B MAO, respectively, in this tissue, whereas the other substrates were metabolized by both forms of MAO. No evidence for any significant metabolism of 5-HT or benzylamine by other amine oxidases was obtained. However, some conditions under which the carbonyl reagents semicarbazide, isoniazid and aminoguanidine may interfere with assays for MAO, without actually affecting enzyme activity directly, are described. Preincubation of kidney mitochondria with histamine resulted in a time- and oxygen-dependent irreversible inhibition of both type A and type B MAO activity; the exact nature of the inhibitory agent and its mode of action remain to be determined.     

Monoamine oxidase (XXXVI). Characteristics of benzylamine oxidase in the dog serum]

Enzymic properties of monoamine oxidase (MAO) in dog serum were studied and the following results were obtained. Some of enzymic properties of MAO in dog serum differed from that of mitochondrial MAO. When dog serum was fractionated by ammonium sulfate, proteins were concentrated in two fractions, such as 25 approximately 33% and 67 approximately 80% of saturated ammonium sulfate fraction, while MAO activity was concentrated in 40 approximately 50% of saturated ammonium sulfate fraction. The reaction rate of MAO in dog serum was found to be proportional to enzyme concentration. The optimum pH of MAO in dog serum was 7.0 which differed from that of MAO in rabbit serum (pH 8.0). Tris-HCl buffer strongly inhibited MAO activity in dog serum. When benzylamine was used as substrate, the highest activity was obtained compared with the other substrate used. The activities with butylamine, amylamine, beta-phenylethylamine and tyramine showed about 30% while tryptamine and serotonin showed 3 approximately 10% compared to that with benzymlamine as substrate. The value of pI50 of catron was about 3 X 10(-6) M and that of harmaline was about 3 X 10(-5) M, but pargyline did not inhibit MAO activity in dog serum at the concentration of 1 X 10(-4) M.     

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.     

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.     

Further Studies on the Ex-vivo Effects of Procarbazine and Monomethylhydrazine on Rat Semicarbazide-sensitive Amine Oxidase and Monoamine Oxidase Activities

Following administration of the anticancer agent, procarbazine, or one of its metabolites, monomethylhydrazine, to rats, activities of monoamine oxidases A and B (MAO A and MAO B) and of semicarbazidesensitive amine oxidase (SSAO) were measured ex-vivo. Both compounds were found to be potent inhibitors of SSAO in tissue homogenates, exhibiting ID50 values in most tissues of approximately 8 mgkg^?1 (procarbazine) and 0.08 mg kg^?1 (monomethylhydrazine). Concurrent dose-dependent inhibition of MAO activities did not occur. However, in liver, potentiation of MAO B activity, to 140% of that in controls, was apparent following monomethyl-hydrazine and this effect was independent of the drug dose. Both compounds produced a dose-dependent potentiation of MAO A in brown adipose tissue, the elevation being more pronounced following monomethylhydrazine, with activity rising to 350% of that in control homogenates. In a parallel in-vitro study, monomethylhydrazine was without effect on MAO A in brown adipose tissue homogenates. By perfusing the SSAO substrate, benzylamine, through the isolated mesenteric arterial bed of the rat, it was found that pretreatment of animals with procarbazine or monomethylhydrazine reduced metabolism of this amine by a similar degree as had been determined ex-vivo in blood vessel homogenates. The results presented suggest that these compounds would be suitable for use as selective inhibitors in pharmacological examinations of SSAO function in isolated tissues and organs.     

Tyramine antagonistic properties of AGN 1135, an irreversible inhibitor of monoamine oxidase type B.

1 The effects of the irreversible monoamine oxidase (MAO) inhibitors, AGN 1133, AGN 1135 and (-)-deprenyl, on tyramine and noradrenaline responses and uptake of [3H]-metaraminol were investigated in the isolated vas deferens of the rat. Uptake of [3H]-metaraminol and [3H]-octopamine was compared in mouse vas deferens. The modification of tyramine and noradrenaline-induced pressor responses by AGN 1133 and AGN 1135 was examined in anaesthetized rats and cats. 2 AGN 1133 (7.5 x 10(-6)M) greatly potentiated responses to tyramine in the rat isolated vas deferens. Both AGN 1135 and (-)-deprenyl inhibited tyramine responses selectively at concentrations above 10(-5)M (which caused almost complete inhibition of MAO types A and B) but tyramine responses were potentiated on washing out the inhibitors. 3 AGN 1135 (10(-4)M) and (-)-deprenyl (10-5)M) inhibited [3H]-metaraminol uptake by about 20% in rat and mouse vas deferens; neither inhibitor affected [3H]-octopamine uptake in mouse vas deferens. Desmethylimipramine (10(-6)M) inhibited amine uptake by more than 70%. 4 AGN 1133 (1.5 mg/kg) potentiated pressor responses to tyramine in rats and cats whereas AGN 1135 (1.5 mg/kg) did not. 5 AGN 1135 possesses tyramine antagonistic activity which is qualitatively similar to that of (-)-deprenyl but which cannot satisfactorily be explained by inhibition of neuronal or granula amine uptake.     

Inhibitory actions of hydralazine upon monoamine oxidizing enzymes in the rat

The inhibition by hydralazine of the clorgyline-resistant amine oxidase (CRAO) and monoamine oxidase (MAO) activities in various rat tissues has been studied. Hydralazine was a potent, time-dependent inhibitor of rat heart CRAO activity in vitro. The inhibition was not reversed by dialysis for 18 hr at 4 degrees, and only partially reversed by dialysis at 37 degrees. Dialysis at 4 degrees in the presence of pyridoxal phosphate (10(-4) M) also did not reverse the inhibition. Ex vivo inhibition of CRAO was found in heart and aorta homogenates in a dose-dependent manner after administration of hydralazine (1-40 mg/kg i.p.) to rats. In contrast, MAO-A activity was unaffected or, in some cases, significantly increased in these tissue homogenates from drug-treated animals. However, in vitro inhibition by hydralazine of both MAO-A and B activities of rat liver mitochondrial fractions was found, and these effects were fully reversible by dialysis for 18 hr at 4 degrees. Inhibition of MAO-A was competitive (Ki of 2.5 X 10(-6) M), while inhibition of MAO-B showed complex mixed non-competitive kinetics. These results indicate that hydralazine possesses different inhibitory properties towards the various amine oxidases in rat tissues, and these actions are discussed in relation to the clinical use of the drug as an anti-hypertensive agent.     

Limitation of adipose tissue enlargement in rats chronically treated with semicarbazide-sensitive amine oxidase and monoamine oxidase inhibitors

Inhibition of semicarbazide-sensitive amine oxidases (SSAO) and monoamine oxidases (MAO) reduces fat deposition in obese rodents: chronic administration of the SSAO-inhibitor semicarbazide (S) in combination with pargyline (MAO-inhibitor) has been shown to reduce body weight gain in obese Zucker rats, while (E)-2-(4-fluorophenethyl)-3-fluoroallylamine, an SSAO- and MAO-B inhibitor, has been reported to limit weight gain in obese and diabetic mice. Our aim was to state whether such weight gain limitation could occur in non-obese, non-diabetic rats and to extend these observations to other amine oxidase inhibitors. Prolonged treatment of non-obese rats with a high dose of S (900 micromol kg(-1) day(-1)) reduced body weight gain and limited white adipose tissue enlargement. When chronically administered at a threefold lower dose, S also inhibited SSAO activity but not fat depot enlargement, suggesting that effects other than SSAO inhibition were involved in adipose tissue growth retardation. However, combined treatment of this lower dose of S with pargyline inhibited SSAO, MAO, energy intake, weight gain and fat deposition. Adipocytes from treated rats exhibited unchanged insulin responsiveness but impaired antilipolytic responses to amine oxidase substrates. Phenelzine clearly inhibited both MAO and SSAO when tested on adipocytes. Obese rats receiving phenelzine i.p. at 17 micromol kg(-1) day(-1) for 3 weeks, exhibited blunted MAO and SSAO activities in any tested tissue, diminished body weight gain and reduced intra-abdominal adipose tissue. Their adipocytes were less responsive to lipogenesis activation by tyramine or benzylamine. These observations suggest that SSAO inhibition is not sufficient to impair fat deposition. However, combined MAO and SSAO inhibition limits adiposity in non-obese as well as in obese rats.     

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.     

Inhibition of rat fat cell lipolysis by monoamine oxidase and semicarbazide-sensitive amine oxidase substrates

It has been demonstrated that amine oxidase substrates stimulate glucose transport in cardiomyocytes and adipocytes, promote adipogenesis in pre-adipose cell lines and lower blood glucose in diabetic rats. These insulin-like effects are dependent on amine oxidation by semicarbazide-sensitive amine oxidase or by monoamine oxidase. The present study aimed to investigate whether amine oxidase substrates also exhibit another insulin-like property, the inhibition of lipolysis. We therefore tested the influence of tyramine and benzylamine on lipolytic activity in rat adipocytes. These amines did not modify basal lipolysis but dose-dependently counteracted the stimulation induced by lipolytic agents. The response to 10 nM isoprenaline was totally inhibited by tyramine 1 mM. The blockade produced by inhibition of amine oxidase activity or by 1 mM glutathione suggested that the generation of oxidative species, which occurs during amine oxidation, was involved in tyramine antilipolytic effect. Among the products resulting from amine oxidation, only hydrogen peroxide was antilipolytic in a manner that was potentiated by vanadate, as for tyramine or benzylamine. Antilipolytic responses to tyramine and to insulin were sensitive to wortmannin. These data suggest that inhibition of lipolysis is a novel insulin-like effect of amine oxidase substrates which is mediated by hydrogen peroxide generated during amine oxidation.     

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.     

On the presence of a clorgyline resistant benzylamine oxidase activity in mouse kidney

In the crude homogenates of three different strains (C3H, Swiss, C57b) of mouse kidney the exposure to a millimolar concentration of clorgyline reveals a benzylamine oxidative deaminating activity that is due to a clorgyline resistant amine oxidase (CRAO). In the three strains this CRAO activity shows a low sensitivity to semicarbazide and to alpha- amiguanidine. In the C3H mouse kidney the glycoprotein nature of the CRAO was established from affinity chromatography with immobilized concanavalin A. Studies of this activity in the C3H mouse kidney revealed the presence of a reversible inhibitor which is precipitated by ammonium sulphate between 35-55% of saturation. The presence of this inhibitor precludes accurate measurement of the CRAO distribution in the different subcellular fractions. The enzyme purified by affinity chromatography, is semicarbazide insensitive. This is the first observation of the presence of a CRAO-semicarbazide insensitive activity in a tissue. The fact that "original homogenate" and the subcellular fractions show some sensitivity to semicarbazide also indicates that a semicarbazide-sensitive amine oxidase (SSAO) is present in the C3H mouse kidney.     

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.     

Amine oxidase activities in brown adipose tissue of the rat: identification of semicarbazide-sensitive (clorgyline-resistant) activity at the fat cell membrane

Amine oxidase activity, previously described in homogenates of brown adipose tissue of the rat, has now been investigated in preparations of isolated fat cells. It was found that the specific activities of both monoamine oxidase A (MAO) and of the semicarbazide-sensitive clorgyline-resistant amine oxidase (SSAO) were higher in isolated fat cells than in the original whole tissue. Brown adipocytes therefore represent a major source of both these enzymes. In plasma membranes prepared from these isolated brown fat cells by borate extraction there was a similar enrichment of activity of SSAO and of the plasma membrane marker enzyme, phosphodiesterase I. However in preparations of cell membranes made by binding the cells to polycation-coated beads, enrichment of phosphodiesterase I activity was much greater than that of SSAO. It is suggested that the disposition of the enzyme within the cell membrane may account for the discrepancy in these results, i.e. the sidedness of the membrane may be important. Histochemical visualization of enzyme activity in whole tissue at the ultrastructural level was undertaken. Positive staining of mitochondria was achieved in the presence of the MAO substrate, tryptamine. Staining around the edges of the brown fat cells was observed with the SSAO substrates, tyramine and benzylamine. Staining was largely absent when substrate was omitted or after pretreatment with the irreversible SSAO inhibitor, hydralazine and the slowly reversible inhibitor, semicarbazide. It is not definitely proven that this staining represents sites of enzyme activity but the results are consistent with evidence from other studies indicating that SSAO in brown adipose tissue of the rat may be found predominantly at the fat cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Monoamine oxidase activity of macrophages at rest and during phagocytosis

Mouse macrophages contain monoamine oxidase (MAO) A activity and traces of MAO B, as judged by a strong deamination of 5-hydroxytryptamine and tyramine and a marginal one of benzylamine. Significant inhibition of MAO activity occurred in the presence of the specific inhibitors clorgyline and deprenyl. MAO A activity was considerably depressed in phagocytizing cells.