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.     

A sedimentation procedure for the detection of binding to concanavalin A and heparin to lipoproteins.

Specific enzymatic bands in disc gel electrophoresis are generally determined by either of two methods: (i) Gel is sliced and the enzymatic activity is assayed on each slice or (ii) gel is stained histochemically, if the product of the enzymatic reaction and the dye can form an insoluble precipitate, and the activity band is located on the gel by a color band. The former is laborious and often inaccurate in the calculation of electrophoretic mobility. The latter, often nonspecific, is not applicable when the enzymatic product cannot form an insoluble precipitate with the dye. Staining with tetrazolium salt has been widely employed for amine oxidase (1–6). However, this method has limitations: (i) Tetrazolium salt is nonspecific for amine oxidase and may show artifacts (6,7), and (ii) the use of tetrazolium salt is limited only to substrates containing indolamine such as tryptamine or serotonin (8). Other substrates, like benzylamine, the most active substrate for plasma amine oxidase, do not form a color band with tetrazolium salt.This communication reports a simple spectrophotometric method for the identification of the enzymatic activity band for amine oxidase on disc gel electrophoresis. Neither slice and assay nor staining is needed. This method may possibly also be used generally for other enzyme systems which have a specific absorption at ultraviolet or visible range.     

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     

Characterization of phosphatidylserine synthase from Saccharomyces cerevisiae and a mutant defective in the enzyme

The membrane fraction of exponentially growing cells of Saccharomyces cerevisiae was found to exhibit phosphatidylserine synthase activity. The enzyme was solubilized by Triton X-100 and chromatographed on a Sepharose 6B column. The enzyme had a pH optimum between 8.0 and 8.5. The apparent Km values for CDPdiacylglycerol and L-serine were 0.12 and 13 mM, respectively. Triton X-100 stimulated the enzyme. Mg2+ or Mn2+ was required for the activity. Ca2+ was inhibitory at relatively low concentrations. The enzyme was highly specific to L-serine. Labeling experiments showed that the enzyme synthesized phosphatidylserine by transferring the phosphatidyl moiety to L-serine. A mutant of S. cerevisiae defective in phosphatidylserine synthase was isolated. The strain required ethanolamine for its growth. Ethanolamine could be substituted by choline or high concentrations of L-serine. The mutant showed normal levels of CDPdiacylglycerol-inositol 3-phosphatidyltransferase and phosphatidylethanolamine methyltransferase activities.     

A phospholipid serine base exchange enzyme

A membrane bound L-serine exchange enzyme which catalyzes the exchange reaction between L-serine and phospholipid-base was solubilized and separated from the ethanolamine-exchange enzyme by Sepharose 4B and DEAE-cellulose column chromatography. The separated fraction was purified approximately 37-fold with a yield of 2--5%. This fraction did not possess ethanolamine or choline exchange activity. The optimal pH was approx. 8.0, the incorporation rate of L-serine into phospholipid was linear up to 20 min incubation time and the activity was maximum at 10 mM CaCl2. The calculated Km value for L-serine was 0.4 mM. Ethanolamine phospholipid was the most effective acceptor for L-serine incorporation, particularly ethanolamine plasmalogen. The Km values obtained were: 0.25 mM for ethanolamine plasmalogen, 0.25mM for pig liver phosphatidylethanolamine and 0.66 mM for egg yolk phosphatidylethanolamine. These observations suggest that the hydrophobic moiety in ethanolamine phospholipid, as well as the base moiety, is important for the affinity of the L-serine exchange enzyme. Neither ethanolamine nor choline inhibited the L-serine exchange activity. There was no detectable conversion of phosphatidylcholine or phosphatidylethanolamine to phosphatidic acid by the partially purified enzyme.     

Inhibition of lentil copper/TPQ amine oxidase by the mechanism-based inhibitor derived from tyramine

Copper amine oxidase from lentil (Lens esculenta) seedlings was shown to catalyze the oxidative deamination of tyramine and three similar aromatic monoamines, benzylamine, phenylethylamine and 4-methoxyphenylethylamine. Tyramine, an important plant intermediate, was found to be both a substrate and an irreversible inhibitor of the enzyme whereas the other amines were not inhibitory. In the course of tyramine oxidation the enzyme gradually became inactivated with the concomitant appearance of a new absorption at 560 nm due to the formation of a stable adduct. Inactivation took place only in the presence of oxygen and was probably due to the reaction of the enzyme with the oxidation product of tyramine, p-hydroxyphenylacetaldehyde. The kinetic data obtained in this study indicate that tyramine represents a new interesting type of physiological mechanism-based inhibitor for plant copper amine oxidases.     

Novel copper amine oxidase activity from rat liver mitochondria matrix

Copper containing amine oxidases (Cu-AO) represent a heterogeneous class of enzymes classified as EC 1.4.3.6. The present study reports preliminary results on the presence of a novel amine oxidase activity in rat liver mitochondria lysates. Such enzymatic activity was found in the soluble mitochondrial fraction, obtained by simple osmotic shock. The mitochondrial amine oxidase was isolated by affinity chromatography on a newly synthesised spermine-Sepharose. SDS-PAGE showed a single band at about 60 kDa. Upon chromatographic purification, the enzymatic activity was very labile. The crude enzyme activity was tested by spectrophotometric measurements, determining hydrogen peroxide production following oxidative deamination of different substrates, such as polyamines (spermine, spermidine, putrescine and cadaverine) and monoamines (dopamine and benzylamine). The activity, observed on polyamines and not on monoamines, was inhibited by semicarbazide and azide, but not by pargyline, clorgyline and l-deprenil. Enzyme specificity was tested on several diamines characterized by different carbon atom chain length in the range 2-6 carbon atoms. The highest activity was found with 1,2-diamino-ethane and the highest affinity with 1,5-diamino-pentane. The above reported results suggest the presence of a novel copper-dependent amine oxidase in liver mitochondria matrix.     

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

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

Monoamine oxidase, an intracellular probe of oxygen pressure in isolated cardiac myocytes

The activity of monamine oxidase, an enzyme located almost exclusively at the outer mitochondrial membrane, toward the substrate phenylethylamine is used to report the oxygen pressure at the outer mitochondrial membrane of intact cardiac myocytes isolated from hearts of adult rats. The rate of substrate oxidation, under the conditions used, follows the Michaelis-Menten relation, and accordingly can be used as a measure of the local chemical activity of dissolved oxygen. The oxygen pressure at the outer mitochondrial membrane of myocytes, at rest and after 2- to 3-fold stimulation of respiratory oxygen consumption, differs from the extracellular oxygen pressure by at most 2 torr. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. At physiologically relevant concentrations of the substrates phenylethylamine and norepinephrine, monoamine oxidase activity is relatively insensitive to extracellular oxygen pressure in the range 155 to 8 torr, suggesting a limited role for regulation of biogenic amine oxidation by oxygen availability.     

Modulation by phospholipids of the activity of monoamine oxidase purified from pig liver

Monoamine oxidase was purified from pig liver mitochondria to homogeneity. The enzyme sample contained a large amount of phospholipids. Depletion of lipids from the enzyme sample resulted in a decrease in its activity, while activity was restored by the binding of the lipid-depleted enzyme to phosphatidylcholine, phosphatidylethanolamine, or mitochondrial lipids. Upon binding the lipid-depleted enzyme to the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), the enzymatic activity toward serotonin was elevated over that of the purified enzyme, but not toward benzylamine, suggesting a change in substrate specificity. Upon lipid depletion, inhibition by deprenyl became weaker, while that by clorgyline became stronger. This alteration was reversed by the binding to lipids. By the binding of the lipid-depleted enzyme to some lipids such as the mixture of phosphatidylcholine and phosphatidylethanolamine (molar ratio, 1 : 1), inhibition by clorgyline became even weaker than for the original enzyme sample.     

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.     

Salt effect on pig kidney diamine oxidase activity

The aim of the present investigation was to study specific effects of ionic strength on the amine oxidase activity. Different patterns were shown in the variation of enzymatic activity, according to the cationic species and the substrates tested (cadaverine and butylamine). Different cations exert specific effects, particularly sodium, which seems to regulate in some way the diamine oxidase activity (Fig. 1 and 2). Involvement of active sites of enzyme interacting with charged molecules is postulated.     

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.     

Preparation of Amine Oxidase from Bovine Serum by Affinity Chromatography on Aminohexyl-Sepharose

Amine oxidase was purified from bovine serum by affinity chromatography on aminohexyl substituted Sepharose. The enzyme was adsorbed on the chromatographic support in a suspension of aminohexyl Sepharose in diluted serum. After thorough washing with buffer, the gel was packed in a column and the enzyme eluted with 10 mM octylamine. Using this procedure it was possible to obtain apparently homogenous amine oxidase in a single-step procedure. The specific enzyme activity was 0.14 μoles benzaldehyde formed per minute at 25°C per mg enzyme protein. Based on the activity of amine oxidase in serum, the yield of enzyme was 64 %.     

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.     

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.     

Multiplicity of monoamine oxidase: inhibition of mitochondrial monoamine oxidase activity by isopropylhydrazide of D,L-serine]

Isopropylhydrazide of D,L-serine (IHS) inhibits by 50% (at 37 degrees for 10 min) deamination of serotonin or beta-phenylethylamine by monoamine oxidases from bovine brain stem mitochondrial membranes at the 2.6 X X 10(-5) M or 9 X 10(-5) M, respectively. In order to inhibit by 50% the deamination of tyramine under the same conditions a considerably lower (2.5 X X 10(-6) M) concentration of IHS is required. Kinetic studies of inhibition of enzymatic deamination of all the three biogenic monoamines by IHS showed that the irreversible blocking of the monoamine oxidase activity is preceeded by formation of dissociating enzyme-inhibitor complexes. Values of the dissociation constants of these complexes measured (at 37 degrees) with serotonin, phenylethylamine or tyramine as substrates for estimation of the residual monoamine oxidase activity are 0.47; 0.13 or 0.023 mM, respectively. Significant differences are also found between thermodynamic and activation parameters characterizing both both steps of interaction between IHS and the monoamine oxidases of mitochondrial membranes in the experiments with serotonin, phenylethylamine or tyramine as substrates. The data obtained suggest the existence of different monoamine oxidases (or their active sites) catalyzing oxidative deamination of serotonin, phenylethylamine or tyramine in the fragments of mitochondrial membranes from bovine brain stem.     

Purification and characterization of sweet potato cytochrome c oxidase.

Sweet potato cytochrome c oxidase (EC 1.9.3.1) was purified 45-fold with respect to its specific activity, with a high recovery by solubilization of the enzyme from the submitochondrial particles with deoxycholate, diethylaminoethyl-cellulose column chromatography, and fractionation with ammonium sulfate. Impurities, if any, could be removed by sucrose density gradient centrifugation of the purified enzyme preparation, although a considerable inactivation of the enzyme took place during centrifugation. The purified enzyme contained approximately 12 nmol of heme a per milligram of protein and about 2.5% phospholipid. The cytochrome c oxidase consisted of at least five polypeptides with molecular weights of 39,000, 33,500, 26,000, 20,000, and 5700, as determined by polyacrylamide gel electrophoresis of the purified enzyme preparation in the presence of sodium dodecyl sulfate and urea. Phosphatidylcholine and phosphatidylethanolamine stimulated the activity over 3-fold. The optimal pH of the purified enzyme was 7.0 to 7.5 in the presence of phosphatidylcholine (egg yolk or soybean) and pH 6.5 in the presence of phosphatidylethanolamine.     

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.