Enzymatic characterization of an amine oxidase from Arthrobacter sp. used to measure phosphatidylethanolamine

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS-PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.     

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.     

OCTOPAMINE IN MAMMALIAN BRAIN: RAPID POST MORTEM INCREASE AND EFFECTS OF DRUGS

A modified enzyme radiochemical assay for octopamine, based upon the N-methylation of octopamine by the enzyme phenylethanolamine N-methyl transferase (S-Adenosyl-1-methionine: phenylcthanolamine N-methyl transferase EC 2.1.28), has been developed. [³H]Methyl-S-adenosyl-l- methionine was used as methyl donor, and the reaction products separated by thin-layer chromatography prior to liquid scintillation counting. The method had a sensitivity of about 100 pg, and was suitable for the measurement of endogenous octopamine levels in mammalian brain. Although the method could be used for the determination of phenylethanolamine with similar sensitivity, concentrations of this amine in brain were too low for routine measurement. Octopamine levels in the brains of a number of mammalian species were determined using this procedure. Concentrations of the amine in mouse brain were lower in animals killed by rapid freczing than in animals killed by decapitation; a further increase in brain octopamine took place post-mortem. Brain octopamine was increased following treatment with MAO inhibitors, p-chlorophenylalanine, phenylalanine, tyrosine or phenylethylamine. The effects of tyrosine and phenylethylamine were greatly increased by pretreatment with a monoamine oxidase inhibitor. The antidepressants imipramine and iprindole gave rise to increased brain octopamine concentrations, possibly through an effect upon monoamine oxidase. Administration of chlordiazepoxide chlorpromazine, thyroxine, or reserpine had no effect upon brain octopamine.     

Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Amine Oxidases of Microorganisms

An improved purification procedure is described for preparation of crystalline amine oxidase in higher yields from the mycelial extract of Aspergillus niger. The amine oxidase was accumulated in mycelia of the fungus when it was grown in a medium containing n-butylamine as a sole nitrogen source. The n-butylamine was fed from the alkali tank of a pH stat connected with the jar fermenter, to maintain pH of the culture medium at 5.0~5.5 during the incubation.

The amine oxidase was shown to be inhibited by guanidine hydrochloride and dissociation of the enzyme occurred when it was dialyzed against 6 m guanidine hydrochloride containing 0.1 m mercaptoethanol. The native enzyme of its molecular weight of 252,000 dissociated into three subunits of each molecular weight of 85,000 during the dialysis.

Pyridoxal derivatives were detected in digest of the native enzyme by proteolysis followed by acid hydrolysis. The derivatives showed a biological activity to support the growth of Saccharomyces carlsbergensis. Fluorescence property of these derivatives rather resembled to that of the known pyridoxal derivatives. A ¹⁴C-labelled protein was prepared from a solution containing the enzyme and ethylamine-1-¹⁴C, through the reductive action of sodium borohydride. On acid hydrolysis of the protein, a radioactive compound was obtained that has chromatographic and fluorescence properties of pyridoxylethylamine.     

Cytokinins as Inhibitors of Plant Amine Oxidase

Abstract

The interaction of pea seedling amine oxidase with cytokinins was examined to probe a possible connection between cytokinin oxidase and amine oxidase by determining whether cytokinins are substrates or inhibitors of the latter. Kinetic measurements suggest that cytokinins are weak competitive inhibitors of amine oxidase while their behaviour as substrates was not observed. The absence of enzymatic activity with cytokinins as substrates denies the identity or even any similarity of these two enzymes which was previously considered [Hare, P.D. and van Staden, J. (1994) J. Physiol. Plant., 91, 128]. From the values of the inhibition constants obtained it seems unlikely that cytokinins take part in the regulation of amine oxidase activity in vivo. Their inhibitory effect on amine oxidase may be similar to that of some alkaloids studied earlier.     

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

Oxidation of amines by yeasts grown on 1-aminoalkanes or putrescine as the sole source of carbon,nitrogen and energy

The maximum growth rate of Trichosporon cutaneum CBS 8111 in chemostat cultures was 0.185 h^-1 on ethylamine and 0.21 h^-1 on butylamine, that of Candida famata CBS 8109 was 0.32 h^-1 on putrescine.The amine oxidation pattern of the ascomycetous strains studied, viz. Candida famata CBS 8109, Stephanoascus ciferrii CBS 4856 and Trichosporon adeninovorans CBS 8244 was independent of the amine that had been used as the growth substrate. It resembled that of benzylamine/putrescine oxidase found in other ascomycetous yeasts. However, differences in pH optimum and substrate specificity were observed between the amine-oxidizing systems of these three species.The amine oxidation pattern of cell-free extracts of Trichosporon cutaneum CBS 8111 varied with the amine that was used as growth substrate. The enzyme system produced by Cryptococcus laurentii CBS 7140 failed to oxidize isobutylamine and benzylamine, and showed a high pH optimum.The synthesis of amine oxidase in the four yeast strains studied was not repressed by ammonium chloride and was weakly repressed by glucose but was strongly repressed if both compounds were present in the growth medium.     

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.     

Tissue-specific and substrate-specific detection of aldehyde and pyridoxal oxidase in larval and imaginal tissues of Drosophila melanogaster

The substrate specificities of aldehyde and pyridoxal oxidases in Drosophila melanogaster have been determined with a variety of aliphatic and aromatic aldehydes. This analysis has led to the discovery that 2,4,5-trimethoxy-benzaldehyde is a specific substrate for pyridoxal oxidase, as based on the histochemical distribution of oxidase activity, the absence of enzymatic activity in the lpo ¹strains, and the dosage dependence on the number of lpo ⁺genes present. The tissue-specific localization of aldehyde oxidase (AO) and pyridoxal oxidase (PO) in the larval and adult structures showed that AO was present in all the major internal organs of the larvae and adults, including brain, imaginal discs, Malpighian tubules, digestive system, and reproductive structures. Pyridoxal oxidase is present in many of the same structures which possess AO, but is missing from the cardia, crop, imaginal discs, ovarian follicle cells, paragonia, pericardial cells, and wreath cells. The only structure which possesses PO but lacks AO is the larval salivary gland. These histochemical differences in AO and PO distribution were also confirmed by enzymatic analysis of the activities present in homogenates of ovaries, paragonia, and salivary glands. The general pattern of enzyme expression appears to be established during embryogenesis and maintained throughout the life of the individual.This work was supported by NIH Grants AG01975 and GM27866.This paper is dedicated to Professor Donald F. Poulson, Yale University, a pioneer in Drosophila developmental genetics.     

2-Arylthiomorpholine derivatives as potent and selective monoamine oxidase B inhibitors

2-Arylthiomorpholine and 2-arylthiomorpholin-5-one derivatives, designed as rigid and/or non-basic phenylethylamine analogues, were evaluated as rat and human monoamine oxidase inhibitors. Molecular docking provided insight into the binding mode of these inhibitors and rationalized their different potencies. Making the phenylethylamine scaffold rigid by fixing the amine chain in an extended six-membered ring conformation increased MAO-B (but not MAO-A) inhibitory activity relative to the more flexible α-methylated derivative. The presence of a basic nitrogen atom is not a prerequisite in either MAO-A or MAO-B. The best K_i values were in the 10^?8 M range, with selectivities towards human MAO-B exceeding 2000-fold.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

Immobilized yeast cells with methanol oxidase activity: Preparation and enzymatic properties

Cells form the yeast Hansenula polymorpha (ATCC 26012) were successfully immobilized by entrapment in a polyacrylamide gel. The resulting gel showed high methanol oxidase activity especially after treatment with a detergent (CTAB). The enzymatic properties of the gel-entrapped cell were not very different from that of the soluble enzyme except that no inhibition was observed at high methanol concentration. In continuous reactors, the gel-entrapped cells showed a much higher stability than other enzyme preparations. The inactivation mechanism was investigated and proved to be the oxidation of essential SH group(s) of the methanol oxidase molecule by hydrogen peroxide. Treatment with β-mercaptoethanol prevented inactivation or regenerated activity.     

Characterization of the amine oxidase involved in the growth of Trichosporon cutaneum X4 on ethylamine as source of carbon,nitrogen and energy

The amine oxidase from Trichosporon cutaneum X₄ grown on ethylamine as carbon, nitrogen and energy source was purified to near homogeneity. The purified enzyme showed the highest resistance to heat of any amine oxidase hitherto characterized from a yeast (half-life at 62°C, 14 min). Measurement of kinetic parameters as a function of carbon chain length showed results typical of a benzylamine oxidase. Both non-denaturing- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed multiple bands, and dimethyl suberimidate cross-linking studies revealed that the enzyme consisted of multimers of two polypeptide chains of M_r respectively 19,000 and 26,000. The smallest structure to show activity probably contained two of each kind of subunit.Abbreviation SDS sodium dodecyl sulphate     

The IAA-oxidase system of citrus roots

Summary The activity of indoleacetic acid oxidase in citrus plants (roots of mature trees and roots and stems of seedlings) was demonstrated. The interrelationships between 2,4-dichlorophenol, Mn⁺⁺ and the enzymatic activity were examined. The presence of Mn⁺⁺ was not found necessary, even in dialyzed enzyme reaction mixtures. However, Mn⁺⁺ promoted the enzymatic activity at the very high concentration of 10^-1M. The inhibitory effect induced by excessive amounts of 2,4-dichlorophenol (10^-3 M) was removed in the presence of Mn⁺⁺.The enzyme showed a specificity to IAA as compared with other synthetic auxins in a wheat coleoptile bioassay. Moreover, the enzyme acted selectively on some endogenous auxins found in citrus tissues.The existence of endogenous inhibitors in stems and cofactors in roots was also demonstrated.This study is part of a project carried out under grants authorized by Public Law 480 for the U.S. Department of Agriculture (10-CR-22, FG-Is-136). The financial support provided is gratefully acknowledged.     

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml⁻¹. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml⁻¹). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani. This revised version was published online in November 2006 with corrections to the Cover Date.     

Temporal control of urate oxidase activity during development of the third-instar larva of Drosophila: The role of 20-hydroxyecdysone

The tissue-specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third-instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20-hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third-instar larvae is demonstrated using the temperature sensitive mutant ecd¹ which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation and thus remains a third-instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd¹ larvae at 19°C is identical to that of the wild type. However, at 29°C ecd¹ third-instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd¹ larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20-hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS-polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti-Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.     

Isolation and characterization of phenylethylamine and phenylethanolamine from human brain

The Presence of endogenous 2-phenylethylamine in mammalian tissues has long been suspected, in view of the fact that L-phenylanine, a substrate for L-aromatic amino acid decarboxylase (Lovenberg , Weissbach and Udenfriend , 1962), is found in substantial amounts in many neural and non-neural tissues. It has been difficult to demonstrate the presence of phenylethylamine in tissues of untreated animals because this amine is an excellent substrate for monoamine oxidase (Mantegazza and Riva , 1963). Using paper chromatography and electrophoresis, Nakajima , Kakimoto and Sano (1964) tentatively identified phenylethylamine in many organs of animals pretreated with monoamine oxidase inhibitors. Phenylethylamine exerts, in animals pretreated with such inhibitors, behavioural stimulant effects similar to those induced by amphetamine (Mantegazza and Riva , 1963). These effects may in part be attributable to catecholamine release (Fuxe , Grobecker and Jonsson , 1967) and partly to a direct effect exerted by phenylethylamine itself (Fischer , Ludmer and Sabelli , 1967; Giardina , Pedemonte and Sabelli , 1972). The brain content of phenylethylamine in mice (Mosnaim and Sabelli , 1971), rabbits (Sabelli , Giardina , Mosnaim and Inwang , 1972) and rats (Fischer , Spatz , Heller and Reggiani , 1972) is increased by antidepressive treatments (imipramine, monoamine oxidase inhibitors, electroshock) and reduced by reserpine. The urinary excretion of phenylethylamine is decreased in depressed patients (Fischer , Heller and Miró , 1968; Boulton and Milward , 1971; Inwang , Sugerman , Mosnaim and Sabelli , 1972; Fischer et al., 1972). However, the presence of phenylethylamine in brain has not yet been conclusively demonstrated because the analytical procedures used in the above-mentioned investigations were not sufficiently specific. In the present study we isolated and identified, by a number of analytical procedures, phenylethylamine and its metabolite 2-hydroxy-2-phenylethylamine (phenylethanolamine) from human brain. Molinoff , Landsberg and Axelrod (1969) have shown by enzymatic methods the formation of phenylethanolamine following the administration of phenylethylamine.     

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.     

Evidence of parietal amine oxidase activity in Solanum torvum Sw. stem calli after Ralstonia solanacearum inoculation

Calli induced from Solanum torvum stem explants were inoculated with Ralstonia solanacearum under partial vacuum. All calli showed a hypersensitive response after infiltration. Furthermore, amine oxidase activity with aldehyde and H₂O₂ production was detected in semi-purified cell walls of calli infiltrated by the bacteria. Due to its preferential affinity for monoamines, this enzyme is supposed to have monoamine oxidase-like (MAO-like) activity. Moreover, the presence of hydroxyl radicals in the aromatic cycle alters the oxidative deamination kinetics of potential substrates. Indeed, the oxidation of dopamine (+2, OH) was shown to be faster than that of tyramine (+1, OH), which in turn was faster than that of phenylethylamine (0, OH). The MAO-like catalytic activity was significantly inhibited by some reducing agents such as sodium bisulphite and cysteine, and also by tryptamine under anaerobiosis. This latter result suggested that the prosthetic group of the MAO-like enzyme could be a tyrosine-derived 6-hydroxytopaquinone structure. Finally, the sigmoid kinetics of the MAO-like enzyme in semi-purified cell walls did not correspond to that expected for a purified MAO, suggesting that the kinetics were affected by some factors present in cell walls.