Distribution of membrane-bound monoamine oxidase in bacteria.

The distribution of membrane-bound monoamine oxidase in 30 strains of various bacteria was studied. Monoamine oxidase was determined by using an ammonia-selective electrode; analyses were sensitive and easy to perform. The enzyme was found in some strains of the family Enterobacteriaceae, such as Klebsiella, Enterobacter, Escherichia, Salmonella, Serratia, and Proteus. Among strains of other families of bacteria tested, only Pseudomonas aeruginosa IFO 3901, Micrococcus luteus IFO 12708, and Brevibacterium ammoniagenes IAM 1641 had monoamine oxidase activity. In all of these bacteria except B. ammoniagenes, monoamine oxidase was induced by tyramine and was highly specific for tyramine, octopamine, dopamine, and norepinephrine. The enzyme in two strains oxidized histamine or benzylamine. Correlations between the distributions of membrane-bound monoamine oxidase and arylsulfatase synthesized in the presence of tyramine were discussed.     

Monoamine oxidase in adult Hymenolepis diminuta (Cestoda).

A membrane-bound monoamine oxidase (EC 1.4.3.4) was demonstrated in homogenates of Hymenolepis diminuta. The enzyme oxidized a variety of biologically active amines (in decreasing order: dopamine, adrenaline, noradrenaline, tryptamine, tyramine, octopamine), there was, however, no activity with 5-hydroxytryptamine or benzylamine. No diamine oxidase (EC 1.4.3.6.) could be detected in H. diminuta (using histamine, cadaverine or putrescine as substrates). The monoamine oxidase from H. diminuta was not inhibited by azide, hydroxylamine or semicarbazide, but was inhibited by cupferron, alpha-alpha dipyridyl and iodoacetamide, and by the specific monoamine oxidase inhibitors pargyline, nialamide and iproniazid. Several anthelmintics were also found to be inhibitors of monoamine oxidase. The possible roles of monoamine oxidase in H. diminuta are discussed.     

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.     

Gene cloning of the maoA gene and overproduction of a soluble monoamine oxidase from Klebsiella aerogenes

Summary We cloned the structural gene for monoamine oxidase (maoA) from Klebsiella aerogenes into a pKI212 vector in an maoA mutant strain of K. aerogenes. Deletion analysis and complementation tests of the recombinant plasmid showed that the maoA gene was located entirely within a 4.1-kb segment. In an maoA mutant strain harbouring the cloned maoA gene, synthesis of monoamine oxidase was induced by addition of tyramine and related compounds. Transfer of a plasmid containing the maoA gene into a monoamine oxidase-producing strain of K. aerogenes W70 resulted in about a 30- to 40-fold increase in total production of the enzyme. When cells of K. aerogenes carrying the plasmid containing the maoA gene were grown with tyramine, more than 85% of the monoamine oxidase was produced in soluble form, whereas the parent strain W70 produced most monoamine oxidase as the membrane-bound form. Offprint requests to: Y. Murooka     

Catabolite repression and derepression of arylsulfatase synthesis in Klebsiella aerogenes

When a mutant (Mao(-)) of Klebsiella aerogenes lacking an enzyme for tyramine degradation (monoamine oxidase) was grown with d-xylose as a carbon source, arylsulfatase was repressed by inorganic sulfate and repression was relieved by tyramine. When the cells were grown on glucose, tyramine failed to derepress the arylsulfatase synthesis. When grown with methionine as the sole sulfur source, the enzyme was synthesized irrespective of the carbon source used. Addition of cyclic adenosine monophosphate overcame the catabolite repression of synthesis of the derepressed enzyme caused by tyramine. Uptake of tyramine was not affected by the carbon source. We isolated a mutant strain in which derepression of arylsulfatase synthesis by tyramine occurred even in the presence of glucose and inorganic sulfate. This strain also produced beta-galactosidase in the presence of an inducer and glucose. These results, and those on other mutant strains in which tyramine cannot derepress enzyme synthesis, strongly suggest that a protein factor regulated by catabolite repression is involved in the derepression of arylsulfatase synthesis by tyramine.     

Functional groups of mitochondrial monoamine oxidase]

Functional groups of mitochondrial monoamine oxidase critical for monoamine oxidase activity were investigated by chemical modification of highly purified monoamine oxidase preparations from pig liver by specific inhibitors. The substrate and inhibitory properties of synthesized derivatives of beta-phenylethylamine, containing various acylating and alkylating groups in the p-position of the benzene ring, were studied. It was shown that 4-carbmethoxy-beta-phenylethylamine (I) is readily deaminated by monoamine oxidase, whereas 4-O-acetyl-beta-phenylethylamine (II) is not affected by the enzyme. 4-O-acetyl-beta-phenylethylamine (II) and 4-ethyl-O-chloroacethyl phenol (III) inhibit deamination of tyramine, 4-amino-beta-phenylethylamine, beta-phenylethylamine, 4-chloro-beta-phenylethylamine and serotonin in different degrees. The kinetic studies demonstrated that this inhibition is probably due to the acylating properties of the compounds obtained. Selectivity in inhibition may be accounted for by acylation of the group of monoamine oxidase which is located in the nearest proximity to the nucleophynoamine oxidase which is located in the nearest proximity to the nucleophylic site of monoamine oxidase active centre important for binding of tyramine. This group is neither the imidazole group of histidyl, nor the SH-group of cysteinyl residues of monoamine oxidase protein molecule. Its nature is discussed in the light of the data obtained.     

Purification, cloning, and three-dimensional structure prediction of Micrococcus luteus FAD-containing tyramine oxidase

The FAD-containing tyramine oxidase enzyme and gene from the Gram (+) bacterium Micrococcus luteus were isolated, and computer prediction was used to propose a preliminary 3D model of the protein. A 2.8-kb Sau3AI fragment containing the structural gene of tyramine oxidase was cloned from a M. luteus genomic DNA library. The 1332 bp gene encodes a protein of 443 amino acids, with a calculated molecular mass of 49.1 kDa. The enzyme was found to be a homodimer with a molecular weight of 49,000. It oxidizes tyramine, adrenaline, 3-hydroxytyramine, dopamine, and noradrenaline, and was reversibly inhibited by FAD-containing monoamine oxidase A and B specific inhibitors. Sequence comparison show that tyramine oxidase is smaller than other FAD-amine oxidases but that it contains well-conserved amino acid residues reported in all other FAD-amine oxidases. A hypothetical three-dimensional structure of tyramine oxidase has also been proposed based on secondary structure predictions, threading, and comparative modeling.     

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.     

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β-d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K_m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.     

Effect of solubilization by methylethyl ketone of mitochondrial monoamine oxidase of the rat liver on the kinetic properties of the enzyme

The values of Km and V for serotonin, tyramine and beta-phenylethylamine deamination by solubilized and partially purified preparations of monoamine oxidase (MAO) from rat liver were determined. As a result of MAO solubilization by methylethylketone, 14% of activity localized in the mitochondria passed into solution. Subsequent chromatography on AH-Sepharose 4B resulted in 27-fold purification of the enzyme with a 9% yield. In experiments with membrane-bound and partially purified MAO, the Km and V values were shown to increase non-competitively with a rise in O2 concentration. In contrast with intact mitochondria, the use of partially purified MAO preparations led to a loss of the enzyme sensitivity to O2 depending on the nature of the amine. The dependence of kinetic properties of MAO on the lipid environment of mitochondrial membranes is discussed.     

Regulation of derepressed synthesis of arylsulfatase by tyramine oxidase in Salmonella typhimurium.

The participation of tyramine oxidase in the regulation of arylsulfatase synthesis in Salmonella typhimurium was studied. Arylsulfatase synthesis was repressed by inorganic sulfate, cysteine, methionine, or taurine. This repression was relieved by tyramine, octopamine, or dopamine, which induced tyramine oxidase synthesis, although the level of arylsulfatase activity was very low. The induction of tyramine oxidase and derepression of arylsulfatase by tyramine were strongly inhibited by glucose and ammonium chloride, and the repression of both enzymes was relieved by use of xylose as a carbon source after consumption of glucose or by use of tyramine as the sole source of nitrogen, irrespective of the carbon source used. The initial rates of tyramine uptake by cells grown with glucose and xylose were similar. Results with tyramine oxidase-constitutive mutants showed that constitutive expression of the tyramine oxidase gene resulted in derepression of arylsulfatase synthesis in the absence of tyramine. Thus, catabolite and ammonium repressions of arylsulfatase synthesis and the induction of the enzyme by tyramine seem to reflect the levels of tyramine oxidase synthesis. These results in S. typhimurium support our previous finding that the specific regulation system of arylsulfatase synthesis by tyramine oxidase is conserved in enteric bacteria.     

Regulation of tyramine oxidase synthesis in Klebsiella aerogenes.

Tyramine oxidase in Klebsiella aerogenes is highly specific for tyramine, dopamine, octopamine, and norepinephrine, and its synthesis is induced specifically by these compounds. The enzyme is present in a membrane-bound form. The Km value for tyramine is 9 X 10(-4) M. Tyramine oxidase synthesis was subjected to catabolite repression by glucose in the presence of ammonium salts. Addition of cyclic adenosine 3',5'-monophosphate (cAMP) overcame the catabolite repression. A mutant strain, K711, which can produce a high level of beta-galactosidase in the presence of glucose and ammonium chloride, can also synthesize tyramine oxidase and histidase in the presence of inducer in glucose ammonium medium. Catabolite repression of tyramine oxidase synthesis was relieved when the cells were grown under conditions of nitrogen limitation, whereas beta-galactosidase was strongly repressed under these conditions. A cAMP-requiring mutant, MK54, synthesized tyramine oxidase rapidly when tyramine was used as the sole source of nitrogen in the absence of cAMP. However, a glutamine synthetase-constitutive mutant, MK94, failed to synthesize tyramine oxidase in the presence of glucose and ammonium chloride, although it synthesized histidase rapidly under these conditions. These results suggest that catabolite repression of tyramine oxidase synthesis in K. aerogenes is regulated by the intracellular level of cAMP and an unknown cytoplasmic factor that acts independently of cAMP and is formed under conditions of nitrogen limitation.     

The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria.

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.     

Monoamine oxidase electrode in freshness testing of meat

Monoamine oxidase (monoamine: oxygen oxidoreductase, EC 1.4.3.4 from Aspergillus niger and beef plasma) was immobilized in a collagen membrane. An enzyme electrode consisting of a monoamine oxidase - collagen membrane (10 units) and an oxygen electrode was prepared for the determination of monoamines. Monoamines were oxidized to aldehydes by the immobilized enzyme and oxygen consumption was monitored amperometrically by the oxygen electrode. The response time of the electrode was 4 min. The optimum conditions for the enzyme electrode were pH 7.4 and 30°C. A linear relationship was observed between the amine (tyramine) concentration in the range 50–200 μm and the difference in current. No decrease in the output current was observed over an observation period of one week. The difference in current was reproducible with an average relative error of 8%. Monoamines in meat extracts were determined by the enzyme electrode.     

Immunological Studies of Human Monoamine Oxidases

Antisera have been raised against monoamine oxidase preparations from human placenta and platelets. These antisera have been employed to characterize membrane-bound enzyme from a variety of human sources including liver, heart, and brain. The comparisons were based on a displacement radioimmunoassay system with soluble placental monoamine oxidase, previously labelled specifically with [3H]pargyline, as antigen. All forms of enzyme investigated demonstrated immunological cross reaction; however, the placental enzyme appeared to possess determinants not exhibited by the enzyme from the platelets or other tissues examined.     

The nature of the electrophoretically separable multiple forms of rat liver monoamine oxidase

1. Treatment of a partly purified preparation of rat liver monoamine oxidase with the chaotropic agent sodium perchlorate caused the enzyme to migrate as a single band of activity of polyacrylamide-gel electrophoresis, whereas the untreated enzyme separated into a number of bands. 2. Treatment with the chaotropic agent caused no loss of enzyme activity towards benzylamine, dopamine or tyramine. 3. The activities of the untreated preparation towards different substrates were inhibited to different extents by heat treatment and by some inhibitors. No such differences could be detected after the enzyme preparation had been treated with sodium perchlorate. 4. Lipid material, which could be separated by gel filtration, was liberated from the enzyme preparation by sodium perchlorate treatment. 5. The molecular weight of the treated enzyme was found to be 380000+/-38000. 6. Perchlorate treatment altered the solubility of the enzyme. 7. A continuous assay method for monoamine oxidase is described.     

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.     

Multiple substrate determination of monoamine oxidase distribution and iproniazid inhibition in rat brain

Abstract— —A variety of monoamine oxidase substrates (tyramine, dopamine, serotonin, tryptamine) have been used with and without Iproniazid inhibition to evaluate further the extent to which enzyme multiplicity may exist in various regions of rat brain. Levels of monoamine oxidase activity, as measured by ammonia production, were found to vary as a function of both brain area and kind of substrate used, in the absence as well as in the presence of Iproniazid, in vivo and in vitro. Similarity of substrate metabolizing patterns among the different brain areas, however, strongly suggests that only one kind of monoamine oxidase exists in rat brain.     

Liver monoamine oxidase in the obese-hyperglycaemic (ob/ob) mouse.

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.     

STUDIES ON THE MONOAMINE OXIDASE OF MONKEY BRAIN

Abstract— A study of the enzyme monoamine oxidase (MAO) was carried out in the monkey brain. From the monkey brain mitochondrial fraction a lysolecithin-soluble form of the enzyme (MAO_s and an insoluble form (MAO_p) were isolated. The latter required freezing, thawing and sonication for solubilization. Both these forms of MAO had identical electrophoretic mobilities, a pH optimum of 7 and comparable thermal stabilities. The enzyme which could not be solubilized and which remained membrane-bound also gave the same pH optimum of 7 and a similar thermal stability profile. Both MAO_s and MAO_p had comparable K _m values of 2.2 × 10⁻⁵ m and 5.0 ×^105- m respectively when using tyramine as substrate and 7.4 ×⁻⁵ M and 7.7 × 10⁻⁵ m respectively with benzylamine as substrate. The K _m values of the membrane-bound enzyme were 1.0 × 10"⁵m with tyramine as substrate 2.5 × m with benzylarnine as substrate. The MAO inhibitors, tranylcypromine, isocarboxazid and iproniazid inhibited both MAO_s and MAO_p to approximately the same extent. The extent of inhibition of the membrane-bound enzyme however, was relatively different with all three inhibitors. Immunodiffu-sion techniques using anti-MAO_p indicated the immunological identity among MAO_p, MAO_s and the mitochondrial fraction. Substrate specificity and substrate competition experiments as well as the use of the selective inhibitor pargyline indicated the presence of both the 'A' and 'B' type of activity in the MAO isolated from monkey brain.