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.     

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.     

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.     

Monoamine oxidase in adult Ascaridia galli

Monoamine oxidase (MAO), catalysing oxidative deamination of biogenic monoamines, has been detected in adult Ascaridia galli. MAO was present in mitochondria and deaminated noradrenaline at the maximal rate, although serotonin, adrenaline, tyramine and dopamine were also degraded but more slowly. Of the organs studied, the body wall, female reproductive organ and intestine, the body wall (containing neuronal structures) showed highest MAO activity. Km value for chick ascarid mitochondrial MAO using tyramine as substrate was 1.66 X 10(-3) M and it was most active at 2.5 mM tyramine concentration, pH 7.5 and 40 degrees C. MAO of A. galli appeared to be thermolabile as nearly 80% of its activity was lost when the incubation temperature was increased 5 degrees above optimum.     

Alternate substrates of dopamine beta-hydroxylase. I. Kinetic investigations of benzyl cyanides as substrates and inhibitors

A series of benzyl cyanide analogs have been studied as substrates and inhibitors of dopamine beta-hydroxylase to extend our initial report (Baldoni, J. M., and Villafranca, J. J. (1980) J. Biol. Chem. 255, 8987-8990) which showed that p-hydroxybenzyl cyanide was a suicide substrate of dopamine beta-hydroxylase. Thus, the appVmax values for benzyl cyanide analogs decrease in the order p-OH greater than m-OH greater than H much greater than p-OCH3,m-OCH3; the m-OH, m-OCH3 and p-OCH3 analogs are competitive inhibitors versus tyramine in initial velocity studies. The Vmax values for tyramine and p-hydroxybenzyl cyanide are nearly identical at saturating O2 and ascorbate (pH 5.0, 37 degrees C) but the Km for O2 is 0.14 and 2.8 mM, respectively, with tyramine and p-hydroxybenzyl cyanide. Studies of the pH dependence of log V/K for tyramine show two pKa values of 5.2 and 5.8 while for m-hydroxybenzyl cyanide the values are 5.3 and 5.9. The log Vmax profile shows one pKa of 5.9 with tyramine as substrate. Thus, nearly identical enzymic groups are involved in binding and/or catalysis with these two substrates. All the benzyl cyanide analogs are suicide inactivators of dopamine beta-hydroxylase. With m-hydroxybenzyl cyanide, the partition between catalysis and inactivation (kcat/kinact) changed from approximately 600 to approximately 17 as the pH varied from 5.0 to 6.7. The log kinact versus pH profile shows one pKa value of 6.0, suggesting that an enzymic group must be deprotonated for maximal inactivation. Copper was essential for the suicide inactivation of dopamine beta-hydroxylase by benzyl cyanides and kinetic studies of partially inhibited dopamine beta-hydroxylase (approximately 50%) showed that inactive enzyme molecules were completely inactive. The following papers in this series discuss the partial reactivation of suicide-inhibited dopamine beta-hydroxylase and the stoichiometry of inactivation by benzyl cyanide analogs.     

The effects of 5-hydroxytryptophan and 5-hydroxytryptamine on dopamine synthesis and release in rat brain striatal synaptosomes.

The effects of 5-hydroxytryptophan (5-HTP) and serotonin (5-HT) on dopamine synthesis and release in rat brain striatal synaptosomes have been examined and compared to the effects of tyramine and dopamine. Serotonin inhibited dopamine synthesis from tyrosine, with 25% inhibition occurring at 3 μM-5-HT and 60% inhibition at 200 μM. Dopamine synthesis from DOPA was also inhibited by 5-HT, with 30% inhibition occurring at 200 μ. At 200 μM-5-HTP, dopamine synthesis from both tyrosine and DOPA was inhibited about 70%. When just the tyrosine hydroxylation step was measured in the intact synaptosome, 5-HT, 5-HTP, tyramine and dopamine all caused significant inhibition, but only dopamine inhibited soluble tyrosine hydroxylase [L-tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] prepared from lysed synaptosomes. Particulate tyrosine hydroxylase was not inhibited by 10 μM-5-HT, but was about 20% inhibited by 200 μM-5-HT and 5-HTP. At 200 μM both 5-HT and 5-HTP stimulated endogenous dopamine release. These experiments suggest that exposure of dopaminergic neurons to 5-HT or 5-HTP leads to an inhibition of dopamine synthesis, mediated in part by an intraneuronal displacement of dopamine from vesicle storage sites, leading to an increase in dopamine-induced feedback inhibition of tyrosine hydroxylase, and in part by a direct inhibition of DOPA decarboxylation.     

Reduction of membrane-bound dopamine beta-hydroxylase from the cytoplasmic surface of the chromaffin-granule membrane.

Resealed bovine chromaffin-granule 'ghosts' were used for assaying the membrane-bound form of dopamine beta-hydroxylase. Hydroxylation of the substrate tyramine is dependent on its accumulation within the 'ghosts', where the active site of the enzyme is located. Free tyramine in the medium is at a low concentration, low ionic strength and a relatively high pH (7.0), so that even in the presence of a reducing agent (co-reductant) the unaccumulated amine is hydroxylated at a negligible rate. 'Ghosts' contain an endogenous co-reductant, which is shown to be catecholamine remaining in the membrane itself after granule lysis. Catecholamine that is free in solution in the medium or in the interior of the 'ghosts' is not effective as co-reductant, nor is ascorbate, in contrast with the situation with soluble dopamine beta-hydroxylase. Ferrocyanide is an active co-reductant, however, giving a hydroxylation rate approximately equal to the tyramine accumulation rate: it does not enter the 'ghosts', nor does the enzyme appear to utilize ferrocyanide sealed inside the 'ghosts'. A mechanism must therefore exist for transferring electrons across the membrane from the cytoplasmic surface to the matrix surface. NADH is not an electron donor for the enzyme, nor is cytochrome b-561 involved.     

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.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Initial catabolism of aromatic biogenic amines by Pseudomonas aeruginosa PAO: pathway description, mapping of mutations, and cloning of essential genes

Pseudomonas aeruginosa PAO1 was able to utilize several aromatic biogenic amines as sole sources of carbon or nitrogen. These included the phenethylamines tyramine and dopamine and the phenethanolamines octopamine, synephrine, and norepinephrine. Initial catabolism of the phenethylamines was mediated by a membrane-bound tyramine dehydrogenase which produced 4-hydroxyphenylacetaldehyde (4HPAL) with tyramine as the substrate. The enzyme was induced by growth with both classes of amines. Initial catabolism of octopamine (except when present as the sole source of carbon and nitrogen) was mediated by a soluble enzyme with activity against the phenethanolamines but not against tyramine or dopamine. The product of the reaction with octopamine as substrate was also 4HPAL. Addition of NAD to reaction mixtures yielded 4-hydroxyphenylacetic acid and NADH. These activities, octopamine hydrolyase and 4-HPAL dehydrogenase (measured as a combined activity, OCAH-4HPALDH), were only induced by growth with phenethanolamines. However, the combined activities were not observed in extracts from cells grown with octopamine as the sole source of carbon and nitrogen, suggesting that an alternate pathway is used under this growth condition. Two independently isolated mutant strains were unable to utilize tyramine as a sole source of carbon or nitrogen. These mutants were also unable to utilize dopamine but grew at wild-type rates on the phenethanolamines. The mutations were mapped at about 70 min on the PAO1 chromosome with the chromosome-mobilizing plasmid R68.45, and both were linked to the catA1, mtu-9002, tyu-9009, and puuE mutations. DNA complementing both of the mutations was cloned on a single BamHI fragment approximately 13.8 kilobase pairs in length. Analysis of a subcloned fragment showed that the two mutations were in different genes.     

Use of alternate substrates to probe the order of substrate addition to dopamine beta-hydroxylase

In order to determine the order of substrate binding to dopamine beta-hydroxylase during catalysis, the effect of alternate substrates upon kinetic parameters was examined. The V/K value for ascorbate was unchanged when tyramine, phenylpropylamine, p-Cl-phenethylamine, p-CH3O-phenethylamine, or phenethylamine was the hydroxylated substrate. The V/K values for tyramine and oxygen were similarly unchanged when ferrocyanide was used as the reductant in place of ascorbate. In order to use ferrocyanide as reductant it was necessary to include copper to alleviate the substrate inhibition seen with this substrate. The pattern of substrate inhibition observed with ferrocyanide was consistent with a small amount of free cyanide present in the ferrocyanide. With ferrocyanide as reductant and [2,2-2H2]tyramine as substrate, there was a measurable isotope effect on the V/K value for oxygen, but none on the values of Vmax or V/K for tyramine. These results are consistent with a ping-pong mechanism in which tyramine binds to the enzyme after the release of oxidized ascorbate. Subsequently, oxygen binds to form a ternary complex.     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.     

Inhibition of Escherichia coli fructose-1,6-bisphosphatase by fructose 2,6-bisphosphate

Fructose 2,6-bisphosphate, a potent inhibitor of fructose-1,6-bisphosphatases, was found to be an inhibitor of the Escherichia coli enzyme. The substrate saturation curves in the presence of inhibitor were sigmoidal and the inhibition was much stronger at low than at high substrate concentrations. At a substrate concentration of 20 μM, 50% inhibition was observed at 4.8 μM fructose 2,6-bisphosphate. Escherichia coli fructose-1,6-bisphosphatase was inhibited by AMP (K_j = 16 μM) and phosphoenolpyruvate caused release of AMP inhibition. However, neither AMP inhibition nor its release by phosphoenolpyruvate was affected by the presence of fructose 2,6-bisphosphate. The results obtained, together with previous observations, provide further evidence for the fructose 2,6-bisphosphate-fructose-1,6-bisphosphatase active site interaction.     

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.     

Correlation of copper valency with product formation in single turnovers of dopamine beta-monooxygenase

Chemical- and freeze-quench EPR techniques have allowed single-turnover studies of the copper-containing enzyme dopamine beta-monooxygenase. Reduction of enzyme by a stoichiometric amount of ascorbate followed by rapid mixing with tyramine leads to oxidation of bound copper and formation of hydroxylated product in the expected 2:1 ratio. The tyramine dependence of single turnovers yields a limiting rate of 82 +/- 9 s-1 and Km of 3 +/- 1 mM, in agreement with kinetic modeling based on steady-state parameters. Together these results show that the reduced enzyme is a catalytically competent species, with bound copper acting as the sole reservoir of reducing equivalents. The correlation of copper oxidation and substrate hydroxylation rules out significant antiferromagnetic spin coupling in the enzyme-product complex. Since the enzyme-product complex's Cu2+ EPR signal is absent in the transient approach to the steady state [Brenner, M. C., Murray, C. J., & Klinman, J. P. (1989) Biochemistry (preceding paper in this issue)], this result implies that ascorbate reduces copper in the enzyme-product complex. These findings have important consequences for catalysis and active site structure in dopamine beta-monooxygenase.     

Purification and characterization of deacetylipecoside synthase from Alangium lamarckii Thw

Deacetylipecoside synthase (DIS), the enzyme catalyzing the condensation of dopamine and secologanin to form the (R)-epimer of deacetylipecoside, has been purified 570-fold from the leaves of Alangium lamarckii and partially characterized. The isolated enzyme is a single polypeptide with Mr 30,000, and has a pH optimum at 7.5 and a temperature optimum at 45 degrees C. The apparent Km values for dopamine and secologanin are 0.7 and 0.9 mM, respectively. DIS exhibits high substrate specificity toward dopamine, whereas neither tyramine nor tryptamine are utilized. The enzyme activity is not inhibited by its substrate dopamine, but is inhibited by alangimakine and dehydroalangimakine with similar I50 values of 10 microM. DIS presumably provides (R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that also possess an (R)-configuration at the same chiral center.     

Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

Tyramine,as an Active Factor for the Production of Phenolsulphatase by Aerobacter aerogenes

An active factor for the production of phenolsulphatase by some strains of Aerobacter aerogenes was separated as pure crystals from a commercial peptone preparation by ion exchange chromatographic techniques. Properties of this substance were studied, and it was found to be identical with tyramine. Even when pure tyramine in a concentration of 1 × 10^?5M was added to the culture medium, its effect on the level of enzymatic activity could be observed. In addition to tyramine, hordenine was also found effective but this, only in the case of A. aerogenes strain 9621. This specific activity of tyramine appears to be an induction of enzyme production.     

Properties of an enzyme from bananas (Musa sapientum) which hydroxylates tyramine to dopamine

An enzyme system isolated from the pulp of banana fruit (Musa sapientum) was partially purified and characterized. The enzyme was capable of catalysing the hydroxylation of the monophenol, tyramine, to the diphenol, dopamine (3,4-dihydroxyphenylethylamine). Unlike some tyrosinases, the reaction was not stimulated by catalytic amounts of diphenolic reaction product. Ascorbic acid, however, reduced the initial lag period in the oxidation of tyramine, stimulated the reaction rate and promoted the accumulation of dopamine during the first few minutes of the reaction. The hydroxylation of tyramine was apparently dependent upon molecular oxygen. On the basis of these observations it is tentatively suggested that the enzyme is a tyramine hydroxylase which may be responsible for the formation of dopamine in the banana.     

Effect of butaclamol enantiomers on hypothalamic tyrosine hydroxylase in the rat brain

The authors demonstrate stereospecificity of the action of butaclamol enantiomers on substrate inhibition of hypothalamic tyrosine hydroxylase (TH) and regulation of the tyrosine hydroxylase response by the presynaptic membrane (presynaptic receptors) of rat hypothalamus synaptosomes under membrane activation with dopamine. The effect of (+)-butaclamol on the substrate inhibition of TH was noticeable at a concentration of 10(-8)M, reaching a maximum at 10(-5)M. (-)-Butaclamol administered at the same concentrations did not influence the substrate inhibition of the enzyme. (+)-Butaclamol added to the incubation medium containing hypothalamic synaptosomes concurrently with dopamine (10(-5)M) completely blocked the regulatory action of the latter on TH, with this action mediated via presynaptic receptors. (-)-Butaclamol (10(-5)M) antagonized the action of dopamine under the same conditions. The data obtained indicate high stereo-specificity of butaclamol enantiomers as regards their effect on presynaptic regulation of TH, suggesting that elimination of the substrate inhibition of hypothalamic TH is a stereoselective effect of neuroleptics and can be a prognostically important criterion in the appraisal of compounds with potential neuroleptic activity.