Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent K_m values ranged from 1.1 ± 10^?5m for tryptamine to 2.5 ± 10^?4m for dopamine; (2) substrate specificity from V_max values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.     

Monoamine oxidase activity measurements using radioactive substrates

The use of Amberlite CG-50, Dowex 50 and solvent extraction for separation of the oxidation products of the biogenic amines are compared, and measurements of monoamine oxidase activity using ¹⁴C-labeled biogenic amines are described. K_m data for tyramine, dopamine, tryptamine, and serotonin for monoamine oxidase activity of rabbit brain mitochondria are reported. Rates of product formation from [¹⁴C]tyramine are compared with polarographic measurements of oxygen utilization using purified MAO and intact mitochondria from rabbit liver and brain. Difficulties in comparative measurements of monoamine oxidase activity and some reasons for wide variations in published data are discussed.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: ¹⁴C-labeled tryptamine, dopamine and kynuramine. With dopamin as substrate, the enzyme activity showed decline during 24 and 48 h starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decreased after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Brain region differences and some characteristics of monoamine oxidase type a and b activities in the vervet monkey

Monoamine oxidase in the vervet monkey showed greater variations in activity in six brain regions when tyramine or phenylethylamine was used as the substrate (3.8- to 4.1-fold differences) than when serotonin was the substrate (1.8-fold differences). With phenylethylamine and tyramine as substrates, the highest MAO specific activities were found in the hypothalamus and the lowest in the cerebellum and cortex. With serotonin as the substrate, the highest specific activities were in the mesencephalon and cortex. The inhibition of tyramine deamination by clorgyline and deprenyl yielded biphasic plots indicative of the presence of MAO-A and MAO-B enzyme forms in the vervet brain. On the basis of these inhibitor curves, the vervet brain could be estimated to contain approximately 85% MAO-B and 15% MAO-A, in contrast to rat brain which contains 45% MAO-B and 55% MAO-A. The inhibition of serotonin deamination by deprenyl in vervet brain yielded a biphasic plot, suggesting that some serotonin deamination in the vervet is accomplished by the MAO-B enzyme form. Estimations of the relative amounts of MAO-A and MAO-B based on inhibitor curves or based on substrate ratios yielded proportionate results which were in close agreement across the different brain regions, supporting the validity of these approaches to estimating MAO-A and MAO-B activities.     

Comparative Study of Catalytic Properties of Monoamine Oxidases of the Liver of the Squid Todarodes pacificus and of the Liver of the Wistar Rat

A comparative study of substrate specificity of monoamine oxidase (MAO) in mitochondria of liver of the Pacific squid Todarodes pacificus and of Wistar rats is carried out. It is revealed that the squid liver MAO, unlike the rat liver MAO, is capable of deaminating not only tyramine, serotonin, and benzylamine, but also histamine. The squid liver MAO activity in relation to all studied substrates is approximately 10 times lower, while the sorption ability, several tens times lower, than the rat liver MAO. Semicarbazide, a classic inhibitor of diamine oxidase, at a concentration 1 × 10^–2 M did not inhibit the catalytic activity of both studied enzymes. The specificity of action of an irreversible inhibitor, proflavine, is established, which was seen at deamination of various substrates by the squid liver MAO to the greater degree, than by the rat liver MAO. The values of the bimolecular rate constant of the irreversible inhibition (k _II) by proflavine were 2.5–20-fold higher (depending on substrate) in the case of the squid liver MAO, than of the rat liver MAO. A suggestion is put forward about the probable presence of several centers of substrate binding in the enzyme of the studied marine invertebrate, like in the mammalian enzyme.     

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.     

Monoamine Oxidase Activities in Catfish (Parasilurus Asotus) Tissues

Abstract

The substrate- and inhibitor-related characteristics of monoamine oxidase (MAO) were studied for catfish brain and liver. The kinetic constants for MAO in both tissues were determined using 5-hydroxytryptamine (5-HT), tyramine and β-phenylethylamine (PEA) as substrates. For both tissues, the V_max values were highest with 5-HT and lowest with PEA. The K_m value for the brain was highest with 5-HT, followed by tyramine and PEA; but for the liver its value was highest with PEA, followed by 5-HT and tyramine, although all values were in the same order of magnitude. The inhibition of MAO by clorgyline and deprenyl by use of 5-HT, tyramine and PEA as substrates showed that the MAO-A inhibitor clorgyline was more effective than the MAO-B inhibitor deprenyl for both catfish tissues; a single form was present since inhibition by clorgyline or deprenyl with 1000 μM PEA showed single phase sigmoid curves. It is concluded that catfish brain and liver contain a single form of MAO, relatively similar to mammalian MAO-A.     

Multiple catalytic sites of rat brain mitochondrial monoamine oxidase

We compared the inhibitory and catalytic effects of various monoamines on forms A and B of monoamine oxidase (MAO) on mitochondrial preparations from rat brain in mixed substrate experiments. MAO activity was determined by a radioisotopic assay. MAO showed lower K_m values for tryptamine and β-phenylethylamine than for tyramine and serotonin. The K_m values of the untreated preparation for tyramine, tryptamine, and β-phenylethylamine obtained were the same as those of the form B enzyme and the K_m value for serotonin was the same as that of the form A enzyme. Tyramine and tryptamine were competitive inhibitors of serotonin oxidation and β-phenylethylamine did not bind with form A enzyme or inhibit the oxidation of serotonin, while tyramine and tryptamine were competitive inhibitors of β-phenylethylamine oxidation. Although serotonin was not oxidized by form B enzyme, serotonin was a competitive inhibitor of β-phenylethylamine oxidation. It is suggested that rat brain mitochondrial MAO is characterized by two kinds of binding sites.     

Localization and characterization of phenol sulfotransferase in human platelets

Phenol sulfotransferase was localized as a soluble enzyme in platelets from human blood. The enzyme was found to esterify a variety of endogenous phenolic biogenic amines including tyramine, dopamine, norepinephrine and 5-hydroxytryptamine as well as phenol. Of the substrates tested dopamine was found to be most rapidly conjugated when present at a concentration of 30 μM while tyramine was found to be the best substrate at a concentration of 100 μM. The Km value for tyramine was 59 μM and tyramine concentrations of 400 μM or greater resulted in apparent substrate inhibition. The possible clinical implications of of these findings are discussed.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats.

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: 14C-labeled tryptamine, dopamine and kynuramine. With dopamine as substrate, the enzyme activity showed decline during 24 and 48 h of starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decrease after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Sulfation of Dopamine and Other Biogenic Amines by Human Brain Phenol Sulfotransferase

Abstract: Phenol sulfotransferase was isolated in 100,000g supernatant fractions prepared from postmortem samples of human brain. Since phenol sulfotransferase (PST) has been shown to conjugate the amine neurotransmit-ters in vivo , the abilities of eight different biogenic amines and structurally related compounds to act as substrates for PST were studied. These experiments demonstrate that at a concentration of 20 μM, dopamine (DA) was the best substrate examined and was followed in decreasing order of activity by 3-methoxytyramine (3-MT), tyramine, norepinephrine, 3-methoxy-4-hydroxyphenylethyleneglycol, octopamine, 5-hydroxytryptamine and dihydroxyphenylethyleneglycol. At a substrate concentration of 100 /UM the relative order of activity was altered, so that tyramine became the most rapidly conjugated substrate while the activity of DA and 3-MT relative to the other substrates tested was diminished. This change in substrate affinity with differing substrate concentrations can be explained, at least for DA, by the occurrence of apparent substrate inhibition at concentrations above 25 to 30 μM. Using PST isolated in 100,000g supernatant fractions from human brain, the K_m value for DA was found to be 5.0 μM, while the K_m value for the sulfate-donor 3'-phosphoadenosine-5'-phosphosulfate was 0.25 μM. The ratio of 3- O - to 4- O -DA-sulfate formed in vitro by human brain PST was found to be about 4: 1. In addition, both the 3- O - and 4- O -esters were found not to be deaminated by human brain mitochondrial MAO. The relative role of PST with respect to MAO and catechol- O -methyltransferase in the degradation of the biogenic amine neurotransmitters in human brain is discussed.     

Oxidation of beta-phenylethylamine by both types of monoamine oxidase: effects of substrate concentration and pH.

β-Phenylethylamine (PEA) was characterized as substrate for both type A and type B monoamine oxidase (MAO) in rat brain mitochondria at different substrate concentrations and at different pHs of the reaction media. The experiments on sensitivity to clorygline and deprenyl showed that the inhibition patterns with PEA as substrate differed markedly at different substrate concentrations: at 10 μM, PEA acted as a specific substrate for type B MAO, but at 50–1000 μM it became a common substrate for both types of MAO. The inhibition patterns were also affected markedly by a small change in pH of the reaction medium, especially when PEA concentrations were 50 and 100 μM: the change in pH from 7.2 to 7.8 resulted in the incresse in the proportion of type A MAO by 20–30 per cent. To investigate the mechanisms of such changes in substrate specificity of PEA, kinetic analyses were carried out at pH 7.2 and 7.8 with the uninhibited, the clorgyline-treated (type B) and the deprenyl-treated (type A) enzyme. The Lineweaver-Burk plots for the uninhibited MAO showed strong substrate inhibition for both pHs, which is more marked at pH 7.8 than at pH 7.2. Pretreatment of the enzyme with 10^?7 M clorgyline resulted in generally similar K_m values for PEA to those of the uninhibited enzyme, and the substrate inhibition at pH 7.8 was also stronger than that at pH 7.2. After pretreatment with 10^?7 M deprenyl, the K_m values were higher and the V_max values were lower than those of the uninhibited or the clorgyline-treated enzyme; there was no or only slight substrate inhibition in these curves. These results suggest that the remarkable changes in substrate specificity observed at different PEA concentrations and at different pHs may be due to the strong substrate inhibition of type B MAO.     

Monoamine Oxidase and Catechol-O-Methyl Transferase Activity in Tetrahymena*

SYNOPSIS Tetrahymena pyriformis strain HSM was found to have monoamine oxidase (MAO) and a catechol-O-methyl transferase-like (COMT) activity. As in mammalian tissues, the MAO activity is predominantly localized in the mitochondrial pellet and COMT in the cytosol. The COMT-like activity was present in amounts comparable to several mouse tissues and was inhibited by tropolone. MAO activity was much lower than in any of the mouse tissues tested, and its activity varied greatly from preparation to preparation. The substrate preference of Tetrahymena MAO was tryptamine > serotonin > dopamine, and activity increased with increasing pH from pH 6.5 to pH 7.8, as does that of mouse liver MAO. The K_m of Tetrahymena MAO for tryptamine was 4 μM, an order of magnitude lower than that of mouse liver MAO. Sensitivity to inhibition by MAO inhibitors was variable. In some preparations, no inhibition was observed. In others clear inhibition was obtained, harmine and clorgyline being among the most potent inhibitors.     

Effects of tricyclic antidepressants upon human platelet monoamine oxidase

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

Biochemistry and physiology of monoamine oxidase (MAO) activity in the ring dove (Streptopelia risoria). I. Characteristics of MAO activity in vitro

Monoamine oxidase (MAO) activity was measured in ring dove (Streptopelia risoria) tissues using a fluorometric assay with kynuramine as substrate. Harmaline inhibited MAO activity in a time-dependent manner, and preincubation of enzyme with the drug did not affect its activity. Pargyline produced a slow-onsetting inhibition of activity which was enhanced by preincubation of enzyme and inhibitor. Harmaline displayed reversible non-competitive inhibition of MAO activity. Oxygen is also a substrate for dove MAO, and the reaction apparently involves "ping-pong", double-displacement kinetics. Dove MAO activity is temperature-dependent, with an activation energy of 13.1 kcal/mole.     

Characterization of rat pulmonary monoamine oxidase.

The substrate specificities and kinetics of rat lung monoamine oxidase (MAO) have been studied. Utilizing the irreversible MAO inhibitors, clorgyline and deprenyl, rat lung was shown to possess at least two types of MAO, A and B. Tyramine was a substrate for both forms of the enzyme, whereas 5-hydroxytryptamine (5-HT) was a preferred substrate for the A-form. In contrast to most other tissues, 2-phenylethylamine was not solely a B-type substrate for the rat lung MAO and some metabolism by the A-type was apparent $\text{(}\text{B}\text{A}\text{=}\text{80}\text{20}\text{)}$. Using tyramine as substrate the ratio A/B was shown to be $\text{55}\text{45}$. Rat pulmonary MAO-B was inhibited by deprenyl and the kinetics of MAO-A studied. The Km values for the A-form for tyramine, phenylethylamine and 5-HT were relatively similar and were 270, 244 and 170 μM respectively. Whereas, when the A-form was inhibited by clorgyline, the Km values for the B-form were found to differ considerably: 330, 42 and 850 μM for tyramine, phenylethylamine and 5-HT respectively.     

Multiple forms of monoamine oxidase in rabbit platelets.

Rabbit platelets were found to contain both types A and B MAO activities. The specific enzymatic activity of rabbit platelet MAO was higher for the substrate serotonin than for phenylethylamine. The K_m's for rabbit platelet MAO indicated that the MAO-B enzyme was similar to human platelet MAO and that both MAO-A and MAO-B enzymes in the rabbit platelet are similar to the corresponding forms in the rabbit brain. The drugs clorgyline and deprenyl confirmed the existence of types A and B MAO in the platelet and furthermore indicated that the type A form accounted for approximately 90% of the total enzymatic activity. Amitriptyline at low (micromolar) concentrations selectively inhibited MAO-B activity in both rabbit platelets and brain.     

Inhibition of Monoamine Oxidase by 7-Chloro-4-Nitrobenzofurazan

7-Chloro-4-nitrobenzofurazan (NBD-Cl) is a potent inhibitor of both types of monoamine oxidase (MAO). NBD-Cl competitively inhibited the oxidative deamination of kynuramine catalyzed by human placenta MAO-A, the oxidative deamination of benzylamine catalyzed by bovine liver MAO-B, the oxidative deamination of serotonin catalyzed by rat brain MAO-A, and the oxidative deamination of phenylethylamine catalyzed by rat brain MAO-B. In addition, a time-dependent inactivation of MAOs by NBD-Cl has been demonstrated upon incubation of the enzyme preparations with NBD-Cl at pH 9, but not at pH 7.5. The time-dependent inhibition of MAO by NBD-Cl could be prevented by the addition of 4-nitrophenyl azide, an active site-directed label of MAO, during incubation of the enzyme with NBD-Cl. On the basis of these findings, it is suggested that at pH 9, NBD-Cl modifies one (or more) essential lysine residue(s) in the active sites of the two types of MAO.     

Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods

Recently, evidence has been published which suggests that [Husain, M., Edmondson, D. E., & Singer, T.P. (1982) Biochemistry 21, 595-600] monoamine oxidase [amine:oxygen oxidoreductase (MAO), EC 1.4.3.4] deaminates phenylethylamine and benzylamine via two distinct kinetic pathways which involve either binary or ternary complex formation, respectively. These conclusions were drawn largely from stopped-flow kinetic analysis performed on purified enzyme removed from its native membrane and in the presence of the inhibitory detergent Triton X-100. In this study, d-amphetamine and alternative substrates were used as steady-state probes of the kinetics of deamination by the B form of human brain MAO using native membrane-bound enzyme. Initial velocity studies showed mixed-type patterns for amphetamine inhibition of phenylethylamine, tryptamine, and tyramine when either amine or oxygen was the varied substrate. Slope and intercept vs. amphetamine concentration replots were linear in all cases except for phenylethylamine (hyperbolic); Ki values obtained from linear replots of slope or intercept values were comparable. In contrast, amphetamine was a competitive inhibitor of benzylamine deamination when amine concentration was varied and uncompetitive when oxygen concentration was varied; slope and intercept replots were linear for both. When benzylamine was the alternative substrate inhibitor and tyramine and tryptamine deamination was measured, mixed-type inhibition patterns were obtained when either amine or oxygen concentration was varied; replots of slope and intercept were linear in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)