Characteristics of monoamine oxidase activity in brain and other organs of the adult bullfrog, Rana catesbeiana

1. Monoamine oxidase (MAO) activity was measured in brain, liver, kidney and intestine of the adult bullfrog by a fluorometric method. 2. All tissues contained both type A and type B MAO, on the basis of responses to specific inhibitors, but with different ratios in each tissue. 3. MAO activity was optimum at 30 degrees C. However, MAO type B showed greater activity changes related to incubation temperature than did type A. 4. The Michaelis constant (Km) of MAO also varied with temperature, with a nadir around 20 degrees C. The functional significance of this is not clear. 5. Arrhenius plots showed that the activation energy for MAO B was higher than for MAO A.     

Some characteristics of monoamine oxidase activity in tissues of the adult female grassfrog (Rana pipiens)

1. Monoamine oxidase (MAO) activity was measured fluorometrically in tissues of the grass-frog, Rana pipiens. 2. Incubation with specific inhibitors revealed the presence of two forms of MAO, type A and type B, in all tissues examined, though the ratios were different in each tissue. 3. Liver contained the greatest, and oviduct and skeletal muscle the lowest amounts of MAO activity, and brain also contained relatively high MAO levels. 4. The apparent Michaelis constant (Km) was similar in most tissues but was higher in pancreas, muscle and oviduct. Values of Km were lower than those reported for mammalian tissues.     

Monoamine oxidase activity in several tissues of the goldfish. Carassius auratus

1. Monoamine oxidase (MAO) was determined fluorometrically in male goldfish tissues, and the effects of specific inhibitors determined. 2. MAO activity in kidney greater than intestine greater than pancreas greater than brain approximately liver greater than testis. Apparent Michaelis constant (Km) was higher in the first three and lower in the last three tissues. 3. Specific MAO type A inhibitors (harmaline, clorgyline) were much more effective than a type B inhibitor (deprenyl) in reducing MAO activity. 4. Apparently goldfish tissues contain only a single type A-like MAO.     

Differences in the Structure of A and B Forms of Human Monoamine Oxidase

Abstract: [³H]Pargyline-labeled polypeptides associated with the A and B types of monoamine oxidase (MAO) activity in human tissues were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). [³H]Pargyline was bound to MAO A in a crude mitochondrial fraction from the placental trophoblast of a male newborn and to MAO B in blood platelets from the umbilical vein of the same newborn. [³H]Pargyline was also bound to MAO A and B in a crude mitochondrial fraction from cultured skin fibroblasts of a male adult and to MAO B in blood platelets from the same individual. Specific labeling of proteins associated with type A or type B activity in fibroblast cells was achieved by preincubation with selective B or A inhibitors, respectively. For all tissues, SDS-PAGE of [³H]pargyline-bound samples revealed a labeled protein band of apparent molecular weight 63,000 for MAO A and 60,000 for MAO B. When SDS-solubilized, [³H]pargyline-labeled MAO A and B proteins from the same male newborn were subjected to limited proteolysis and one-dimensional peptide mapping in SDS gels, different patterns of [³H]pargyline-labeled peptides were obtained. These findings indicate that distinct enzyme molecules are associated with the A and B types of human MAO activity.     

The characteristics and distribution of monoamine oxidase (MAO) activity in different tissues of the rainbow trout, Salmo gairdneri

Monoamine oxidase (MAO) activity was determined fluorometrically in brain, intestine, kidney and liver tissues of the rainbow trout, Salmo gairdneri. MAO activity was inhibited by various drugs in a concentration-related manner, with single sigmoid inhibition curves, the inhibitors of type A MAO, harmaline and clorgyline being more effective than deprenyl, an inhibitor of type B MAO. Intestine exhibited greatest MAO activity followed by liver and brain with kidney showing least activity. The Michaelis constants (Km) also showed variability between tissues. Inhibition of MAO by harmaline was non-competitive and dependent on the concentration of substrate present.     

Biochemistry and physiology of monoamine oxidase (MAO) activity in the ring dove (Streptopelia risoria). II. Distribution of MAO in different tissues

Monoamine oxidase (MAO) activity was measured fluorometrically in liver, kidney, intestine and brain of adult male and female ring doves. Liver MAO was inhibited in a concentration-related fashion by clorgyline and harmaline (MAO type A inhibitors) where a plateau in the inhibition curve occurred with about 15% activity remaining, and also by the type B inhibitor deprenyl, which produced a plateau when about 85% activity remained. Kidney, intestine and brain MAO were inhibited in a biphasic manner by harmaline. Results with inhibitors suggest that 85% of liver MAO, 86% of kidney MAO, 88% of intestine and 75% of brain MAO is type A. Using 10(-6) M harmaline to differentiate between MAO-A and MAO-B type activities, the apparent maximal velocities (Vmax) and Michaelis constants (Km) were determined in different tissues. Most activity occurred in the intestine, with proportionally lesser amounts of kidney, liver and brain. The majority of MAO present was in the A form. Except for kidney, Km of MAO-B was higher than that of MAO-A. Both MAO-A and -B activities were higher in the intestines of male birds, although sex differences in content and type of MAO activity were not observed in other tissues of the ring dove.     

Distribution of monoamine oxidase activity in tissues of the urodeles Ambystoma tigrinum (tiger salamander) and Necturus maculosus (mudpuppy)

1. Monoamine oxidase (MAO) activity was determined fluorometrically in tissues of adult mudpuppies, and pre- (young) and post- (adult) metamorphic tiger salamanders. 2. From responses to specific inhibitors it was determined that 95% activity was MAO type A in all tissues. 3. In young salamanders MAO activity was greater in brain and intestine of males than of females, and was considerably higher in kidney of both sexes and in intestine of males compared to adults. 4. MAO activity was distributed differently in the mudpuppy compared to the salamander. Intestine and liver contained high activity and brain had relatively little MAO activity compared to salamander. 5. The apparent Michaelis constant of MAO activity in the different groups and tissues was generally similar, suggesting a similarity of the MAO molecule.     

[3H]Harman Binding Experiments. II: Regional and Subcellular Distribution of Specific [3H]Harman Binding and Monoamine Oxidase Subtypes A and B Activity in Marmoset and Rat

[3H]Harman (1-[3H]methyl-beta-carboline) was used in a novel radioligand binding assay to label selectively and with high affinity monoamine oxidase (MAO) type A. The concentration of the enzyme was determined in six CNS regions of the primate species marmoset (Callithrix jacchus) and of the rat: hypothalamus, hippocampus, cerebellum, cerebral cortex, striatum, and spinal cord. The specific [3H]harman binding in the CNS of the marmoset reveals the same pharmacological profile and other characteristics (affinity, saturability, and reversibility) as in the CNS of the rat. The regional distribution of the [3H]harman binding density (Bmax) in the CNS exhibits a distinct pattern in the marmoset and the rat and a 35 (hypothalamus) to 75% (hippocampus) lower Bmax in the marmoset than in the rat. The Bmax values of [3H]harman binding in the CNS of the marmoset and the rat combined as well as those from visceral organs of the rat (liver, heart, lung, thymus, spleen, and kidney) correlated positively and highly significantly with the respective Vmax values of specific MAO activity of the A type but not of the B type, determined with kynuramine as the substrate. In subcellular fractionation experiments with rat cerebral cortex, the highest [3H]harman binding density (Bmax) and MAO-A activity (Vmax) were detected in mitochondrial fractions and severalfold lower values in the synaptosomal membrane fraction. In conclusion, we suggest that [3H]harman binding is a biochemical tool as a selective marker to quantify MAO-A in the CNS of different mammalian species as well as in extraneuronal tissues.     

Differential Expression of Type A and Type B Monoamine Oxidase of Mouse Astrocytes in Primary Cultures

Abstract: Type A and type B monoamine oxidase (MAO) activities were determined in mouse brain and in primary cultures of mouse astrocytes. Thirty-one-day-old astrocyte cultures exhibited predominantly type A MAO activity. In cultures of the same age, treated with 0.25 mM dibutyryl cyclic AMP under the same culturing conditions, 30% type B MAO was expressed, although dibutyryl cyclic AMP up to 1 mM does not affect MAO activity in vitro. The specific activity of type B MAO increased significantly in older cultures, while type A MAO changed only slightly.     

Substrate Selectivity of Type A and Type B Monoamine Oxidase in Rat Brain

Abstract: Use of the irreversible inhibitors clorgyline and deprenyl showed that rat brain mitochondria contain type A and type B monoamine oxidase (MAO). Tyramine is a substrate for both types of MAO, whereas serotonin is a preferential substrate for type A MAO. In contrast to MAO in other tissues, type A MAO in brain tissue oxidizes β-phenylethylamine (PEA) at high concentrations (0.5 and 1.0 mM). The proportions of type A and type B MAO activities in the mitochondria estimated from the double-sigmoidal inhibition curves of tyramine oxidation were about 70:30 irrespective of the concentration of tyramine. With PEA as substrate, the ratios of type A to type B activities were found to increase from low values at low concentrations to about 1 at 0.5-1.0 mM-PEA, and even higher at further increased concentrations of PEA. At very low (0.01 mM) and high (10.0 mM) concentrations of PEA, single-sigmoidal curves were obtained; with the high PEA concentration the activity was highly sensitive to clorgyline, whereas with the low concentration it was highly sensitive to deprenyl. In deprenyl-pretreated mitochondrial preparations, all the remaining activity towards 0.5-1.0 mM-PEA was shown to be highly sensitive to clorgyline, demonstrating that this activity was indeed due to oxidation by type A MAO. The opposite result was obtained with deprenyl as inhibitor of clorgyline-pretreated preparations, demonstrating that PEA at this concentration was also oxidized by type B MAO in rat brain mitochondria. The K₃ values of type A and type B MAO for PEA were significantly different. On Lineweaver-Burk analysis, plots with PEA as substrate for type A MAO in a deprenyl-treated preparation were linear over a wide concentration range, whereas those for type B MAO in a clorgyline-treated preparation were not linear, but showed substrate inhibition at higher concentrations of the substrate. It is concluded from the present findings that the effect of the substrate concentration must be considered in studies on the characteristics of multiple forms of MAO in various organs and species.     

Studies on the pargyline-binding site of different types of monoamine oxidase

[3H]Pargyline has been covalently linked to active sites of both type A and type B monoamine oxidase (MAO) obtained from various tissues. Rat heart and human placenta were chosen to represent predominantly type A MAO, pig and bovine livers to represent type B MAO, and rat liver and brain to represent mixed type A and type B MAO's. The [3H]pargyline-MAO adducts were isolated and hydrolyzed by proteolytic enzymes, and the labelled peptides (pargyline-binding sites) separated and compared by paper chromatography and by paper electrophoresis at various pH values. Only one common pargyline peptide was obtained from all the different MAO's. The alternative A and B sites were assessed after preincubation of rat liver MAO with the selective inhibitors deprenyl (to block the B site) and clorgyline (to block the A site). Following proteolysis of the [3H]pargyline of both type A and type B MAO from this pretreated rat liver, MAO has been purified by a series of chromatographic and electrophoretic procedures. Micro-Edman degradation, followed by dansylation, revealed the amino acid sequence to be Ser-Gly-Gly-Cys(X)-Tyr. It is concluded that the primary structures immediately surrounding the pargyline-binding sites are identical for both type A and type B MAO in these tissues.     

Characterization of monoamine oxidase isoforms in human islets of Langerhans.

In this paper, we describe the characterization of the expression of monoamine oxidase (MAO) in whole pancreas and in isolated islets of Langerhans from human. Classical monamine oxidase activity assays reveal that both isoforms A & B are present in human pancreas. Two complementary approaches indicated that both MAO A and B are expressed in isolated islet: RT-PCR using specific primers revealed amplification products with the expected size for MAO-A and MAO-B: two peptides corresponding to MAO A (approximately 61 kDa) and B (approximately 55 kDa) were detected using a polyclonal anti MAO-A/MAO-B antiserum. Western blotting and subsequent densitometric analysis indicate that whole and endocrine pancreas express the two isoforms with different relative proportions. Islets appear to express almost twice as much MAO protein as whole pancreas, in near equal proportions of the two isoforms, whereas whole pancreas expresses more MAO-A than the B isoform. The expression of MAO A and B in islets could be the first step toward the characterization of the functional properties of these enzymes in the endocrine pancreas.     

Inhibition of monoamine oxidase by 3,4-dihydroxyphenyl L-alanine and its analogues

L-3,4-Dihydroxyphenylalanine (DOPA) was found to inhibit type A monoamine oxidase in human placental mitochondria. The inhibition proved to be noncompetitive with the substrate, kynuramine, and the inhibition was completely reversible. D-DOPA was found to inhibit monoamine oxidase in the same way, and the apparent Ki values of L- and D-DOPA were obtained to be 154 microM and 133 microM, respectively. L-alpha-Methyl-DOPA was found to inhibit the MAO activity competitively with the substrate, but studies with other analogues of DOPA revealed that the inhibition required an amino and a carboxyl group at alpha-position. The substitution of a hydroxy group at 3 or 4 position of catechol ring with a methoxy group was found to abolish the inhibition of the MAO activity. In addition to type A MAO in human liver and placental mitochondria, type B MAO in liver mitochondria was inhibited by L-DOPA, but type B MAO was less sensitive to L-DOPA. These results were discussed in terms of its possible regulation of the level of biogenic amines in the brain.     

Expression of A and B Types of Monoamine Oxidase in Differentiated Neuroblastoma Hybrid Cells

The total activities of monoamine oxidase (MAO) and the ratio of type B/type A activities were determined in mouse neuroblastoma N1E-115 cells, and in NX31T and NG108-15 hybrid cells derived from mouse neuroblastoma X rat sympathetic ganglion hybrid or mouse neuroblastoma X rat glioma hybrid cells. N1E-115 and NX31T cells possessed type A activities exclusively, although NG108-15 cells showed both type A (65-90%) and type B (10-35%) MAO activities. The activity of type A MAO in NX31T and N1E-115 cells was relatively constant during culturing periods in the presence or absence of dibutyryl cyclic AMP (Bt2cAMP), whereas total MAO activity and the ratio of type B MAO/type A MAO in NG108-15 cells increased as a function of culture periods. Prostaglandin E1 (PGE1) and theophylline, the best known combination to increase intracellular cyclic AMP content of NG108-15 cells, caused similar increases of MAO and of the type B/type A ratio in NG108-15 cells. The results suggest that MAO activity and expression of MAO B activity are regulated in NG108-15 cells in a cyclic AMP-dependent manner.     

Monoamine Oxidases A and B as Components of a Membrane Complex

Abstract: The activities of monoamine oxidase A (MAO A) and monoamine oxidase B (MAO B) represent two independent types of substrate binding site, as indicated by experiments with selective inhibitors and also by substrate competition. We have tried to determine whether A and B active sites of human brain and liver MAO are located on physically separable enzyme forms or as subunits in large membrane-bound complexes. MAO was extracted from several sources by a procedure that was designed to give solubilized enzyme in high-speed supernatants, with ratios of MAO A/MAO B activities similar to those in initial crude homogenates. This solubilized enzyme gave gel filtration profiles that suggested the presence of large molecular complexes. Affinity binding experiments indicated that both MAO A and B activities may occur on the same complexes in tissues that initially contain both activities. These complexes were broken down to enzymatically active subunits by treatment with either low concentrations of sodium dodecyl sulfate, with phospholipase A₂, or with a combination of both agents. Results of this study support a concept of MAO as part of a membrane unit in which A and B are two distinct enzymes embedded in a phospholipid structure. The enzymatic activity of MAO A is critically dependent on associated phospholipids, whereas that of MAO B is not.     

Assignment of A and B Types of Monoamine Oxidase in NCB20 Hybrid Cells to Those of the Parental Cells by Peptide Mapping

The structures of [3H]pargyline-labeled, flavin-containing polypeptides of monoamine oxidase (MAO) from hybrid NCB20 cells, and their parental cells, A/J mouse brain cells and Chinese hamster brain cells, were analyzed and compared by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and limited proteolysis and one-dimensional peptide mapping in SDS gels. After preincubation of mitochondrial preparations with deprenyl or clorgyline, the flavin-containing polypeptide of type A or type B MAO was selectively labeled with [3H]pargyline. SDS-PAGE of [3H]pargyline-labeled mitochondrial samples revealed that the polypeptide with apparent Mr of 62,000 was associated with type A activity in the three types of cells, and that the polypeptide with apparent Mr of 61,000 or 58,000 was associated with type B activity in Chinese hamster brain cells and NCB20 cells or A/J mouse brain cells, respectively. Chymotrypsin digestion of the [3H]pargyline-labeled polypeptides and the peptide mapping in SDS gels from A/J mouse and Chinese hamster brain cells produced identical map patterns between the two type A MAOs, almost the same map patterns (with the exception of one additional peptide fragment) between the two type B MAOs, and different map patterns between type A and type B MAOs. The results of identical treatments of the [3H]pargyline-labeled polypeptides of MAOs in NCB20 cells showed that type A and type B MAO in NCB20 cells were similar to type A MAO of A/J mouse and Chinese hamster brain cells and to type B MAO of Chinese hamster brain cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

2-Naphthylamine, a compound found in cigarette smoke, decreases both monoamine oxidase A and B catalytic activity

Cigarette smokers exhibit a lower monoamine oxidase (MAO; EC 1.4.3.4) activity than nonsmokers. MAO is located in the outer membrane of mitochondria and exists as two isoenzymes, MAO A and B. MAO A prefers 5-hydroxytryptamine (serotonin), and MAO B prefers phenylethylamine (PEA) as substrate. Dopamine is a substrate for both forms. 2-Naphthylamine is a carcinogen found in high concentrations in cigarette smoke. The results of this study show that 2-naphthylamine has the ability to inhibit mouse brain MAO A and B in vitro by mixed type inhibition (competitive and non-competitive). The Ki for MAO A was determined to be 52.0 microM and for MAO B 40.2 microM. The inhibitory effect of 2-naphthylamine on both MAO A and B catalytic activity, supports the hypothesis that smoking decreases MAO activity in vivo, instead that smokers with lower MAO activity are more prone to become a smoker.     

Ganglioside GM1 Causes Expression of Type B Monoamine Oxidase in a Rat Clonal Pheochromocytoma Cell Line, PC12h

The effects of ganglioside supplementation of culture medium on monoamine oxidase (MAO) type A and B activities in a rat clonal pheochromocytoma cell line, PC12h, were examined. The MAO activity in PC12h cells proved to be mainly due to type A MAO, and type B MAO activity was negligible. After supplementation of the culture medium with ganglioside GM1, the PC12 cells were found to express type B MAO activity after 4 days of culture, and the amount of type B activity increased with the number of days of culture. After 3 weeks of culture in the presence of GM1, type B activity was about 10% of the total, whereas in control cells type B MAO activity was only about 0.6% of the total. By kinetic analyses of type A and B MAO in PC12h cells after 3 weeks of culture, the increase of type B MAO activity was found to be due to the increase in amount of type B MAO; the Km values were almost the same and only the Vmax values were increased in the cells supplemented with GM1. Among gangliosides tested GM1 was the most effective in causing expression of type B MAO activity, whereas nerve growth factor was not effective. These results suggest that GM1 and other gangliosides may be involved in the expression of type B MAO in nerve cells and in the regulation of levels of the biogenic amines in the brain.     

Through scaffold modification to 3,5-diaryl-4,5-dihydroisoxazoles: new potent and selective inhibitors of monoamine oxidase B

3,5-Diaryl-4,5-dihydroisoxazoles were synthesized and evaluated as monoamine oxidase (MAO) enzyme inhibitors and iron chelators. All compounds exhibited selective inhibitory activity towards the B isoform of MAO in the nanomolar concentration range. The best performing compound was preliminarily evaluated for its ability to bind iron II and III cations, indicating that neither iron II nor iron III is coordinated. The best compounds racemic mixtures were separated and single enantiomers inhibitory activity evaluated. Furthermore, none of the synthesised compounds exhibited activity towards MAO A. Overall, these data support our hypothesis that 3,5-diaryl-4,5-dihydroisoxazoles are promising scaffolds for the design of neuroprotective agents.