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.     

Inhibition of rabbit monoamine oxidase by doxepin and related drugs.

The psychotherapeutic agent, doxepin, inhibits both the B and A forms of rabbit lung mitochondrial monoamine oxidase (MAO). The K_i values for doxepin inhibition of phenylethylamine (PEA) and 5-hydroxytryptamine (5-HT) deamination is 3 × 10⁻⁵ and 2 × 10⁻⁴M, respectively. Doxepin is, thus, similar to other tricyclic antidepressant drugs in that it has a greater affinity for the B form of the rabbit oxidase. The ability of doxepin and imipramine and its mono and didesmethyl derivatives to inhibit rabbit MAO is decreased as the pH is raised above 8.0. However, results suggest that the basicity of the propylamine side chain has little or no influence on the ability of these drugs to inhibit the rabbit oxidase. In addition, it is demonstrated that 7 × 10⁻⁶M doxepin inhibits PEA deamination by human platelet MAO approximately 50%.     

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.     

Substrate-dependent activation energy of the reaction catalyzed by monoamine oxidase

The effect of temperature on the deamination of 5-hydroxytryptamine, tyramine, and phenethylamine by monoamine oxidase (MAO) of human placenta, beef liver, and rat liver has been studied. Both MAO A and MAO B activities are influenced by the lipid-phase transition and, in some cases, another type of transition. The estimates of activation energy (E_act) for the deamination of 5-hydroxytryptamine, phenethylamine, tyramine, dopamine, and pentylamine at 5–20 °C show that a given substrate is associated with a particular value irrespective of the source of MAO acting upon it. The substrate dependence of E_act is explained by the differences in lipophilicity of the various substrates. The interaction of enzyme and the lipids in the environment of its active site would differ with each substrate, and would give rise to different activated complexes, each corresponding to a given substrate. The E_act values are presumably related to these complexes, rather than to enzyme alone.     

5-Hydroxytryptamine metabolism by the nuclear fraction of rat-liver homogenate

1. The metabolism of 5-hydroxy[1′-¹⁴C]tryptamine creatinine sulphate in the nuclear fraction of rat-liver homogenate was studied. In the incubation mixture five metabolites were found. 2. Two metabolites were not radioactive; one of them was identified as 5-hydroxyindole-3-carboxylic acid and the second tentatively as 5-hydroxyindole-3-aldehyde. 3. 5-Hydroxyindol-3-ylacetic acid, 1′-N-acetyl-5-hydroxytryptamine and 5-hydroxytryptophol were not precursors of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid. 4. It was shown that the metabolism of 5-hydroxytryptamine in the nuclear fraction involves monoamine oxidase, the precursor of 5-hydroxyindolealdehyde and 5-hydroxyindolecarboxylic acid being most probably 5-hydroxyindol-3-ylacetaldehyde.     

[3H]HARMALINE AS A SPECIFIC LIGAND OF MAO A—I. PROPERTIES OF THE ACTIVE SITE OF MAO A FROM RAT AND BOVINE BRAINS

A high-affinity (K_d= 5.9 nM) specific binding site for [³H]harmaline was detected in membranes from rat and bovine brains. Studies of the regional and subcellular distributions of this binding indicated its close association with monoamine oxidase type A activity (MAO A) measured with [³H]serotonin ([³H]5-HT) as the substrate. Maximal binding capacity and MAO A activity were found in mitochondrial enriched fractions. Mitochondria of synaptosomal or extra-synaptosomal origin exhibited very similar properties with respect to [³H]harmaline binding characteristics and MAO A activity. Among psychoactive drugs, only monoamine oxidase inhibitors (MAO I) prevented the specific binding of [₃H]harmaline. Logit-log inhibition curves of binding by MAO I gave only one slope which was not significantly different from 1.0, suggesting the existence of only 1 category of specific sites for [³H]harmaline in the membrane preparations from rat and bovine brains. Consistent with the preferential inhibition of MAO A by harmaline, other MAO I of this class, i.e. clorgyline and Lilly 51641, were 10²-2 × 10³ times more efficient than deprenyl and pargyline, two inhibitors of MAO type B, in displacing [³H]harmaline from its specific binding site. K_i and IC₅₀ values for the inhibition of [³H]harmaline binding by MAO I and MAO substrates (tryptamine, 5-HT, norepinephrine) were almost identical with those characterizing their action on MAO A activity with [³H]5-HT as the substrate. In conclusion, the specific binding site for [³H]harmaline exhibited all the expected properties of the active site of MAO A. Like the technique of precipitation with a specific antibody, binding of [³H]harmaline should be of great help for studying the structural characteristics of the active site of MAO A and determining the number of MAO molecules in tissues under various physiological conditions.     

EFFECTS OF LESIONS AND DRUGS ON BRAIN TRYPTAMINE

Abstract— The effects of various drugs and lesions on rat brain 5-hydroxytryptamine and tryptamine were determined. Monoamine oxidase inhibition caused a proportionately greater increase in tryptamine than in 5-hydroxytryptamine, reserpine depleted 5-hydroxytryptamine but had no effect on tryptamine while p -chlorophenylalanine lowered 5-hydroxytryptamine but increased tryptamine. α-Methyl- p -tyrosine reduced striatal dopamine with no effect on either 5-hydroxytryptamine or tryptamine. Increasing brain tryptophan by amphetamine administration. 24 h food deprivation or giving L-tryptophan did not increase brain tryptamine. However a high dose of ^L-tryptophan (100 or 200mg/kg) together with a monoamine oxidase inhibitor caused a proportionately much greater increase in tryptamine than in 5-hydroxytryptamine. Raphe lesions reduced 5-hydroxytryptamine by 64 per cent and tryptamine by only 29 per cent while intraventricular 6-hydroxydopamine lowered striatal dopamine (56 per cent), had no effect on 5-hydroxytryptamine but reduced tryptamine by 24 per cent, suggesting that tryptamine can be formed in both 5-HT and catecholaminergic neurones.
The results are discussed in relation to the formation, distribution, storage and possible transmitter function of tryptamine in rat brain.     

The effects of some antipsychotic drugs, D-amphetamine, and reserpine on the concentration and rate of accumulation of tryptamine and 5-hydroxytryptamine in the mouse striatum

The mouse striatum contains about 2 ng/g of tryptamine and 600 ng/g of 5-hydroxytryptamine. No significant changes in mouse striatal tryptamine were observed after the administration of chlorpromazine, haloperidol, spiperone, or alpha-flupenthixol. The levels of 5-hydroxytryptamine were moderately reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not by haloperidol. The administration of antipsychotic drugs to mice pretreated with a monoamine oxidase inhibitor (pargyline) produced an increase in the rate of accumulation of striatal tryptamine compared with that of pargyline-treated mice. In contrast, the rate of accumulation of 5-hydroxytryptamine after monoamine oxidase inhibition was reduced by chlorpromazine, spiperone, and alpha-flupenthixol but not haloperidol. D-Amphetamine administration did not change either tryptamine or its 5-hydroxyderivative while reserpine increased tryptamine and reduced 5-hydroxytryptamine. The results suggest that changes in striatal tryptamine may be controlled by the availability of tryptophan, the amino acid precursor of tryptamine.     

Early changes in mitochondrial monoamine oxidase activity of rat liver during 2-acetylaminofluorene feeding

The activities of mitochondrial type A and B monoamine oxidase were determined in the liver of rats fed a diet containing 2-acetylaminofluorene (AAF). Three days after the initiation of AAF-feeding, there was a significant decrease of type B monoamine oxidase activity without affect on type A enzyme. The decreased activity of type B monoamine oxidase, which reached a minimum after three weeks, was sustained for as long as AAF-feeding was continued. Sex-related difference in response to AAF was seen in the rat with respect to the onset and the intensity of the decreased type B monoamine oxidase activity, male rats being more sensitive to the carcinogen than female rats. In contrast to the in vivo effect, AAF showed a potent inhibitory effect on type A monoamine oxidase, rather than on type B enzyme, when added in vitro. The pI₅₀ values were estimated to be 7.5 against type A monoamine oxidase and 4.1 against type B enzyme, respectively. The in vitro inhibition of both types of monoamine oxidase by AAF was competitive. The K_i values for AAF were calculated to be 9.51 · 10^?9 M for type A monoamine oxidase and 1.30 · 10^?5 M for type B enzyme, respectively. In accordance with the potent inhibitory effect of AAF on type A monoamine oxidase in vitro, a single administration of the carcinogen, at a dose of 50 mg/kg, resulted in a marked and temporal decrease of the enzyme activity in the mitochondria of male rat liver. Recovery of the decreased type B monoamine oxidase activity was slow, and the enzyme activity did not return to control levels, even if rats were fed the basal diet for 2 or 4 weeks after the cessation of AAF-feeding.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Purification and characterisation of monoamine oxidase from Avena sativa

FAD-containing monoamine oxidase (MAO; EC 1.4.3.4) oxidises monoamines to their corresponding aldehydes, H₂O₂, and NH₃. It has been purified to homogeneity in mammals, but to our knowledge, there have been no reports of the enzyme in plants. MAO activity was detected in Avena sativa seedlings during germination using benzylamine as substrate. The enzyme was purified to homogeneity (as assessed by native PAGE) by Sephadex G-25, DEAE Sephacel, hydroxyapatite, Mono Q, and TSK-GEL column chromatographies. The molecular mass estimated by gel filtration using the TSK-GEL column was 220?kDa. SDS-PAGE yielded four distinct protein bands of 78, 58, 55, and 32?kDa molecular masses. The pI value of the enzyme was 6.3. The enzyme showed high substrate specificity for an endogenous amine, phenethylamine, which was oxidised to phenylacetaldehde, but not for ethylamine, propylamine, butylamine, pentylamine, dopamine, serotonin, tryptamine, or tyramine. The K _m values for benzylamine and phenethylamine were 2.7?×?10^?4 and 7.1?×?10^?4?M, respectively. Enzyme activity was not inhibited by pargyline, clorgyline, semicarbazide, or Na-diethyldithiocarbamate. Benzaldehyde, the product of benzylamine oxidation, exhibited strong competitive inhibition of enzyme activity with a Ki of 3???M. FAD was identified by ODS-column chromatography as an enzyme cofactor. The enzyme contained 2?mol of FAD per 220,000?g of enzyme.     

PROPERTIES OF PARTIALLY PURIFIED PIG BRAIN STEM TRYPTOPHAN HYDROXYLASE

—Tryptophan hydroxylase form pig brain has been purified using a method which involved sonic disintegration of a whole homogenate, ammonium sulphate fractionation, hydroxylapatite fractionation, column chromatography on Sephadex G-100 or G-200 and finally electrophoresis on poly-acrylamide gel. The enzyme was stabilized during purification by tryptophan and dithiothreitol. The partially purified enzyme has a molecular weight of 55,000-60,000 as measured by gel-filtration. The K_m of the soluble partially purified enzyme was 0-4 mm , which differed significantly from that of the particulate enzyme (0·02mm ). Enzyme activity was not stimulated by ferrous ion. However, it was inhibited by the chelating agents 8-hydroxyquinoline, O-phenanthroline and EDTA. In contrast to dopamine, high concentration of tryptophan (10 mm ), 5-hydroxytryptamine, tryptamine and tyramine at 0-5 mm concentration did not inhibit the enzyme in the presence of dimethyltetrahydropterin (DMPH4). A number of monoamine oxidase inhibitors, phenelzine, pheniprazine and chlorgyline at 1 mm strongly inhibit the formation of 5-hydroxytryptamine. Evidence is presented for the presence of an endogenous inhibitor of tryptophan hydroxylase.     

A radiometric assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A radiometric assay for pyridoxamine 5′-phosphate oxidase (pyridoxamine (pyridoxine) 5′-phosphate:O₂ oxidoreductase (deaminating), EC 1.4.3.5) has been developed utilizing N-(5′-phosphopyridoxyl)[³H]tryptamine. This assay is more sensitive than previously used colorimetric and fluorescent assays for this oxidase and furthermore is applicable to erythrocytes. Tritiated substrate is incubated with an enzyme sample in the presence of excess unlabeled truptamine and the radiolabeled tryptamine product is extracted into toluene and quantitated by liquid scintillation counting.     

Developmental changes in the activity and substrate specificities of mouse brain monoamine oxidase

Developmental changes in monoamine oxidase (MAO) activity in the mouse brain were investigated with the substrates -phenylethylamine (PEA), tryptamine, and 5-hydroxytryptamine (5-HT). In the newborn brain, MAO activity towards PEA was found to be much lower than the adult and to be inhibited by clorgyline in a double-sigmoidal fashion. The inhibition curve shifted to a single-sigmoidal pattern with age. MAO activity towards 5-HT as substrate was inhibited by 90% and in a single-sigmoidal manner by clorgyline throughout the postnatal life. Lineweaver-Burk plots with PEA as substrate presented two linear lines (apparentK _m: 28.6 and 4.1 M) for the newborn and one line (apparentK _m: 11.4 M) for the adult, respectively. The plot with highK _m value for the newborn brain disappeared in a clorgyline-treated preparation. These findings suggest that age-dependent alterations in the ratio of MAO-A/MAO-B activity affect the substrate specificity of the enzyme.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

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.     

Biogenic amine degradation by homogenates of the bulb mite,Rhizoglyphus echinopus (Fumouze and Robin) (Acari: Astigmata: Acaridae)

Whole homogenates of bulb mites rapidly metabolized 2-phenylethylamine (PEA) but were appreciably less active against tryptamine, 5-hydroxytryptamine, and dopamine; no degradation of octopamine was detected. The rate of PEA degradation by bulb mites was dependent upon both substrate and homogenate concentrations. PEA degradation was inhibited by pargyline (pI₅₀, 6.7), tranylcypromine (pI_{50 6.2}), and harmaline (pI_{50 4.1}), but not by 5-chloro-2,4-dimethoxyformanilide. These results suggested that PEA metabolism by bulb mite homogenates was catalyzed mainly by Type B monoamine oxidase.Contribution from the Missouri Agricultural Experiment Station, Columbia, MO. Journal Series No. 9777     

Some characteristics of mitochondrial monoamine oxidase activity in eggs of carp (Cyprinus carpio) and rainbow trout (Salmo gairdneri)

1. Monoamine oxidase (MAO) activity towards tryptamine, 5-hydroxytryptamine (5-HT) and phenylethylamine (PEA) has been measured in mitochondria isolated from carp and trout eggs. 2. In carp eggs all the tested substrates are metabolized and the highest affinity is found with tryptamine. In trout eggs a consistent level of MAO activity is obtained using tryptamine. 3. The inhibition dose-response curves of clorgyline and deprenyl indicate that both in carp and trout eggs there is only one form of mitochondrial MAO, distinct from MAO A and B which have been described in vertebrate tissues. 4. Both in carp and trout egg mitochondria a semicarbazide-sensitive amine oxidase is not involved in the deamination of the used substrates. 5. MAO found in carp and trout eggs might be involved in metabolism of some neurotransmitter monoamines during early developmental stages.     

Modulators of monoamine oxidase in plasma

Addition of small amounts of plasma activated the deamination of tryptamine by platelet monoamine oxidase (MAO). At higher concentrations, plasma inhibited the deamination instead. The inhibition was increased with increasing amounts of plasma added. The inhibition was uncompetitive in nature, partially reversed by prior ultrafiltration of the plasma through PM30 membranes and completely reversed by protein precipitation of plasma with perchloric acid. Addition of high amounts of plasma $\text{in}$$\text{vitro}$ also inhibited the activity of bovine striatal MAO. The inhibition of striatal deamination of tryptamine by plasma was noncompetitive in nature, completely reversed by ultrafiltration through PM30 membranes and partially reversed by perchloric acid treatment. The inhibition of striatal deamination of serotonin was noncompetitive in nature, not reversed by ultrafiltration but completely reversed by perchloric acid treatment. The pattern of inhibition of platelet or striatal MAO by plasma was different from that induced by addition of bovine serum albumin (BSA). Low concentrations of BSA added $\text{in}$$\text{vitro}$ activated the deamination of tryptamine or serotonin by platelet or striatal MAO by decreasing the K_m, while higher concentrations also decreased the V_max. The presence of protein, non-albumin circulating modulators of platelet or striatal MAO in plasma is discussed.     

Enzymologic characteristics of monoamine oxidase from liver of the skipjack tuna Katsuwonus pelamis

Substrate and inhibitory specificity of mitochondrial monoamine oxidase (MAO) from liver of skipjack tuna Katsuwonus pelamis was studied. The results of substrate—inhibitory analysis with application of chlorgilin and deprenyl might be indirect proofs of existence of one molecular MAO form in the tuna liver. Studied enzyme, as liver MAO of terrestrial mammals, deaminates tyramine, tryptamine, dopamine, serotonin, noradrenalin, benzylamine, β-phenylethylamine, N-methylhistamine and does not deaminate histamine, is not suppressed by 10 mM semicarbazide. Takrin, acriflavin, proflavin, acridine orange and pyronine G were established to be irreversible inhibitors of middle strength in respect to MAO of tuna liver. The specificity of inhibitors action upon deamination of various substrates was equal.