Rat Liver Mitochondrial Monoamine Oxidase Research Articles

Selective inhibition of bovine plasma amine oxidase by homopropargylamine, a new inactivator motif.

Propargylic and activated allylic amines are known to inactivate the quinone-dependent plasma amine oxidases, possibly through active-site modification by the alpha,beta-unsaturated aldehyde turnover products. Although homopropargylamine (1-amino-3-butyne, 1) is a nonobvious candidate as a mechanism-based inhibitor, 1 was found to be an unusually potent time- and concentration-dependent irreversible inactivator of bovine plasma amine oxidase (BPAO), exhibiting a 30 min IC(50) of 2.9 microM at 30 degrees C ([BPAO] = 1.2 microM). Preserved cofactor redox activity of the denatured inactivated enzyme indicates that inactivation by 1 involves either a cofactor modification that reverses upon enzyme denaturation or a modification of an active-site residue. Because inactivation by 1 may involve enzyme alkylation by the reactive 2,3-butadienal (3) tautomer of the 3-butynal turnover product of 1, aldehyde 3 was prepared and was found to inactivate BPAO, but only at high concentration. In addition, whereas inhibition by 3 was blunted by the presence of mercaptoethanol, no such protection was observed against 1. The amine whose turnover should lead directly to 3 was prepared (1-amino-2,3-butadiene, 4) and was found to be an even more potent inactivator of BPAO than 1, exhibiting a 5 min IC(50) of 1.25 microM. Rat liver mitochondrial monoamine oxidase was also inactivated by 4, as expected, but only very weakly by 1. Potential mechanisms explaining the selective inhibition of BPAO by 1 are discussed.

Inhibition of rat liver mitochondrial monoamine oxidase by hydrazine-thiazole derivatives: structure-activity relationships

The purpose of this research is to study the relationship between chemical structure and inhibitory activity of some hydrazine-thiazole derivatives on rat liver mitochondria monoamine oxidase (MAO). Forty-five compounds belonging to three series of hydrazine-thiazole derivatives, with either alkylic or arylic substituents in the thiazole ring, were tested. The highest inhibitory activity was observed with piperonyl derivatives 25 and 40, which contain a 4-methyl group in the thiazole nucleus. The structure-activity relationship of MAO inhibitors was established in relation to hydrophobic, electronic and steric hindrance parameters. A mechanism of enzyme inhibition was proposed based on the calculation of HOMO energies.

Irreversible inhibition of rat liver mitochondrial MAO A and MAO B by enantiomers of deprenyl and alpha-methylpargyline.

Using rat liver mitochondrial monoamine oxidase (MAO) A and MAO B, the possible influence of stereochemical factors upon the irreversible inhibition by propargylamine derivatives has been studied using the enantiomers of deprenyl and of alpha-methylpargyline. Whether studying the inhibition of MAO A or MAO B, little difference was found among enantiomeric pairs in the first-order rate constant (k2) for formation of the enzyme inhibitor adduct. Similarly, and with the exception of (S)-D-(+)-deprenyl (k2 = 0 or an extremely low value at MAO A), the computed value of k2 for the individual enantiomers showed little variation between MAO A and MAO B. These results suggest that inhibitor selectivity towards a particular form of the enzyme is determined predominantly at the competitive phase of the inhibition.

MPTP and MPTP analogs induced cell death in cultured rat hepatocytes involving the formation of pyridinium metabolites

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) which has been shown to produce a Parkinson-like syndrome in humans and monkeys also causes cell death in cultures of rat hepatocytes. Treatment of cells with MPTP or its metabolite MPP + (1-methyl-4-phenyl pyridinium ion), resulted in leakage of lactic acid dehydrogenase and 14C-labeled adenine nucleotides, as well as marked depletion of ATP and glutathione. Deprenyl, a specific inhibitor of monoamine oxidase-B, the enzyme catalyzing the oxidation of MPTP into MPP +, blocked the lethal effect of MPTP, but gave no protection from MPP +-induced cell death. The 4′-fluoro and 4′-chloro analogs of MPTP evoked toxicities similar to that of the parent compound, while N-butyl-PTP, 4′-amino-MPTP, and 2′-methyl-MPTP were relatively less toxic. N-Acetylamino-MPTP was found virtually nontoxic. The cell death produced by these analogs was also associated with leakage of [ 14C]adenine nucleotides, which is an indicator of loss of ATP from cells. All these compounds except the N-acetylamino analog were converted to corresponding pyridinium metabolites by liver cells when analyzed by high-pressure liquid chromatography and plasma desorption mass spectrometry. MPTP and its analogs also served as substrates for rat liver mitochondrial monoamine oxidase to varying degrees. Toxicity of various analogs, with the noticeable exception of 2′-methyl-MPTP, was inhibited by deprenyl. These findings indicate that the conversion of MPTP and its analogs to corresponding pyridinium metabolites is essential for the expression of toxicity.

Guanabenz as inhibitor of copper-containing amine oxidases.

The effects of guanabenz on some copper containing amine oxidases are described. Guanabenz 'in vitro' inhibits pig plasma benzylamine oxidase with a IC50 M 5.1 +/- 0.8 X 10(-6) M. It also inhibits pig kidney diamine oxidase and rat liver mitochondrial monoamine oxidase at higher concentrations. The significance of this property of guanabenz is discussed.

Stereoselectivity and isoenzyme selectivity of monoamine oxidase inhibitors: Enantiomers of amphetamine, n-methylamphetamine and deprenyl

The enantiomers of amphetamine, N-methylamphetamine and deprenyl were studied, using a solubilised rat liver mitochondrial monoamine oxidase (MAO) preparation, as competitive inhibitors of MAO-A and MAO-B (5-hydroxytryptamine and β-phenylethylamine as substrate respectively). Only in the case of deprenyl enantiomers inhibiting MAO-B was a preference shown towards the [ R]-configuration enantiomer justifying the use of [ R]-(−)-deprenyl (as compared to the racemate) for the specific inhibition of MAO-B. Recalculation of the observed K i values in terms of the base form of the inhibitor indicated that the activity of all enantiomers fell within a narrow, approximately 25-fold range when inhibiting MAO-B. The selectivity of inhibition of MAO-B by [ R]-(−)-deprenyl cannot therefore be attributed to any specific structural features of the MAO-B isoenzyme form but rather to a lack of affinity of this enantiomer towards MAO-A.

The interaction of rat liver mitochondrial monoamine oxidase with clorgyline plus d-amphetamine

The interaction of rat liver mitochondrial monoamine oxidase with clorgyline plus d-amphetamine

The nature of the inhibition of rat liver monoamine oxidase types A and B by the acetylenic inhibitors clorgyline, l-deprenyl and pargyline

The kinetics of inhibition of rat liver mitochondrial monoamine oxidase by clorgyline, l-deprenyl and pargyline are consistent with a mechanism whereby a reversible interaction between the inhibitor and the enzyme active site under conditions of thermodynamic equilibrium is followed by a time-dependent formation of the covalently-bound enzyme-inhibitor adduct. The K i value for the reversible interaction between clorgyline and monoamine oxidase A is about 1000 times lower than that towards the B-form of the enzyme, and this difference is sufficient to account for most, but not all, of the selectivity of the inhibition caused by this compound. The K i value of the monoamine oxidase B selective inhibitor l-deprenyl towards that form of the enzyme is only about 40-fold lower than that towards the A-form. However, in this case, the rate of formation of the irreversible adduct is considerably faster for the B-form than for the A-form and this makes a major contribution to the selectivity of this compound. Pargyline shows a K i value towards monoamine oxidase B that is only 8 times lower than that towards the A-form and in this case the rates of formation of the enzyme-inhibitor adducts are similar. The significance of these results are discussed in terms of the selective inhibition of monoamine oxidase.

Preferential inhibition of the B-form of monoamine oxidase in the liver of rats given 3'-methyl-4-dimethylaminoazobenzene in the diet

A and B-form monoamine oxidase (MAO) activities were measured in the liver of rats maintained with a diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). A-form MAO activity was similar to the control value throughout the feeding periods with serotonin as substrate. In contrast, B-form MAO activity decreased rapidly and the level of MAO activity was maintained at about 30% with β-phenylethylamine (β-PEA) as substrate. 3'-Me-DAB feeding did not cause any changes in MAO activity in the brain of rats. A single administration of 3'-Me-DAB (100 mg/kg p.o.) failed to alter A and B-form MAO activities for up to 4 days after its administration. The mechanism of inhibition of B-form MAO activity in rat liver mitochondria by 3'-Me-DAB was investigated. The inhibition of 3'-Me-DAB of B-form MAO activity, in vitro, was competitive and reversible. There was no difference in the apparent Michaelis constant toward β-PEA between control and 3'-Me-DAB fed rats. B-form MAO in rat liver mitochondria was titrated with (−)deprenyl; this compound is selective to and an irreversible inhibitor of B-form MAO. The content of B-form MAO in liver mitochondria of rats fed 3'-Me-DAB for 3 weeks was decreased to about 60% of the control level.

Concentration dependence of the oxidation of tyramine by the two forms of rat liver mitochondrial monoamine oxidase

The two forms of monoamine oxidase in rat liver mitochondria were shown to have different K m and maximum velocity values with tyramine as the substrate. K m values of 107±15 μM and 579±45μM were determined for the A- and B-form respectively at pH 7.2 in air-saturated buffer. The maximum velocity of the A-form was found to be approximately half of that of the B-form under these conditions. A consequence of these differences is that the ratio of activities of MAO-A:MAO-B determined from clorgyline inhibition curves will be dependent upon the concentration of tyramine used to assay for enzyme activity. As a fixed concentration of tyramine, the height of the plateau in a clorgyline inhibition curve will also be affected by the presence of a selective competitive inhibitor. Procaine, an MAO-A selective competitive inhibitor, was found to increase the K m value of the MAO-A towards tyramine to 505 ± 172 μM and under these conditions the plateau-height of the clorgyline inhibition curve was not significantly affected by variations of the tyramine concentration over a 20-fold range.

Deuterium isotope effects on the enzymatic oxidative deamination of trace amines

The steady-state kinetics of the oxidative deamination of some trace amines [ p-tyramine ( p-TA), m-tyramine ( m-TA) and β-phenylethylamine (PE)] and the same trace amines containing deuterium in their side-chain (i.e. αα-d 2 and ββ-d 2) have been assessed using rat liver mitochondrial monoamine oxidase (MAO) incubated at 37° in 0.05 M phosphate buffer (pH 7.5). In these in vitro reactions, a considerable reduction in deamination occurred when the deuterium substitution was in the α position. In addition, the isotope effect was found to be related to hydroxyl substitution on the phenyl ring. The apparent ( V/ K) H /( V/ K) D ratios were 4.44, 4.24 and 2.06 for p-TA, m-TA and PE respectively. We have confirmed that the cleavage of the C—H bond at the α position is involved in the rate-limiting step of the enzymatic deamination. In the case where the deuterium substitution was in the ββ position, a slight enhancement of deamination occurred with the ( V/ K) H /( V/ K) D ratio becoming 0.89, 0.86 and 0.95, respectively, for p-TA, m-TA and PE. The selective inhibition of the deamination of the αα-deuterated amines by the specific MAO inhibitors clorgyline (type A) and deprenyl (type B) was not different from that of the corresponding non-deuterated trace amines. The isotope effect was found to be somewhat greater at lower temperatures. Using mixed substrates (i.e. trace amine + corresponding deutrated trace amine), the deuterated amines were observed to exhibit a weak inhibitory effect due simply to competition for the same site on the enzyme.

Metabolism of acetylpolyamines by monoamine oxidase, diamine oxidase and polyamine oxidase

N 1-Monoacetylspermine, N 1, N 12-diacetylspermine and N 1-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N 8-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N 1, N 12-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N 1-monoacetylspermine, N 1, N 12-diacetylspermine and N 1-monoacetylspermidine are attacked by polyamine oxidase.

The binding of [3H] pargyline to rat liver mitochondrial monoamine oxidase.

The synthesis and purification of tritium labelled N-desmethylpargyline and pargyline are described. The suitability of these irreversible suicide inhibitors of monoamine oxidase (MAO) as ligands in binding studies to rat liver mitochondrial MAO has been evaluated. [3H] Pargyline was found to be more satisfactory than its N-desmethyl analogue because of its greater potency and lower proportion of non-specific binding. The binding of pargyline reached saturation when about 31 pmol mg protein-1 was bound. It was not possible to explain the time course of the binding by either simple first or second-order kinetics. [3H]-Pargyline is a potentially valuable ligand for the estimation of the concentration of NAO active centres without the need to remove the enzyme from the mitochondrial membrane.

The nature of the substrate-selective interaction between rat liver mitochondrial monoamine oxidase and oxygen

The activity of rat liver monoamine oxidase was increased in an uncompetitive manner with increasing concentrations of oxygen. However, the value of the Michaelis constant for oxygen, estimated from determinations of enzyme activity with 6 oxygen concentrations and 5–6 amine substrate concentrations, was dependent upon the amine substrate used to assay for activity. In an attempt to determine the nature of these differences, the value of the Michaelis constants for oxygen have been related to the k cat (rate constant of the limiting step in the overall enzyme-catalysed reaction) values of the enzyme towards the different amine substrates. The results are consistent with the hypothesis that the two forms of monoamine oxidase in rat liver are not independent enzyme forms, but interact one with the other in some way.

On rat liver mitochondrial monoamine oxidase activity and lipids

Following earlier observations on the retention of 5-hydroxytryptamine oxidizing activity by a purified preparation of monoamine oxidase from rat liver mitochondria, this fraction has been obtained in a water-soluble form by Triton X-100 gradient gel filtration and DEAE-Bio-Gel A chromatography. The soluble fraction appears to depend on Triton X-100 and phospholipids for its activity. The results seem to implicate membrane lipid components in the expression of rat liver mitochondrial monoamine oxidase activity.

The effects of disulfiram on rat liver mitochondrial monoamine oxidase

Monoamine oxidase (MAO) of rat liver mitochondria was found to be inhibited by disulfiram. The inhibition is pH and time dependent: 50% inhibition was observed by 16.5 μM of disulfiram at pH 9.1 after 30 min of preincubation. At pH 7.4 only slight inhibition was produced despite the high concentration of disulfiram (330 μM) and the preincubation period. The inhibition is irreversible and appears to be of mixed type: noncompetitive at low concentration range of the substrate and uncompetitive at high concentration range. Glutathione at twice the concentration of disulfiram abolished the inhibitory effect of the drug. Ethanol, while by itself has only slight effect on MAO activity, enhanced the inhibitory effect of disulfiram at pH 7.4. At pH 9.1, ethanol alone has no effect on MAO; however, it was found to weaken the inhibitory effect of disulfiram.

The effect of tris buffers on rat liver mitochondrial monoamine oxidase.

The effect of tris buffers upon the monoamine oxidase (MAO) activity in rat liver mitochondria has been investigated. Tris buffer was shown to inhibit MAO in a non-competitive manner with a Ki of 15-25 mM. Tyramine, 5-hydroxytryptamine and beta-phenethylamine but not benzylamine oxidations were all inhibited by tris buffer. All inhibitions, except that of 5-HT, were completely reversible. It is suggested that these effects are produced by conformational changes in the structure of the MAO. The significance of these results is discussed with respect to the use of tris buffers in the extraction and estimation of the activity of MAO.

Metabolic interaction of secondary amines and tertiary amino propiophenones with monoamine oxidase systems

The metabolic interaction of three secondary amines and three nitrogen containing metabolites of safrole (tertiary amino propiophenones) with rat liver mitochondrial monoaroine oxidase systems was studied in vitro employing [7- 14C] benzylamine—HCl as a substrate. The two cyclic secondary amines, piperidine and pyrrolidine, showed hyperbolic competitive inhibition while pure competitive inhibition was observed in case of dimethylamine—HCl and three safrole metabolites. Inhibition characteristics for rat liver, kidney and brain mitochondrial monoamine oxidase with two cyclic secondary amines and tertiary amino propiophenones of safrole and elemicin were investigated manometrically using tyramine and serotonin as the substrates.

Substrate specificity of the different forms of monoamine oxidase in rat liver mitochondria

Rat liver mitochondrial monoamine oxidase was inhibited by deprenil (selective inhibitor for the ‘B form’ of monoamine oxidase) to study the ‘A form’ of the enzyme separately. The activity towards serotonin (usually classified as a substrate for the ‘A form’) was estimated in the presence of additional monoamine oxidase substrates. All of the additional substrates investigated inhibited the activity towards serotonin competitively. In the deprenil inhibited preparation all of the residual activity towards β-phenylethylamine (usually classified as a substrate for the ‘B form’) was shown to be sensitive to the ‘A form’ inhibitor clorgyline, indicating that the ‘A form’ was also able to oxidize this substrate. The K m values of the ‘A form’ for serotonin, tyramine and β-phenylethylamine did not differ significantly. When the ‘B form’ of monoamine oxidase was studied after inhibition of the ‘A form’ by clorgyline, all additional substrates investigated were able to inhibit the activity towards β-phenylethylamine in a competitive fashion. All of the remaining activity towards serotonin in the clorgyline inhibited preparation was sensitive to deprinil. Thus the ‘B form’ also appears to be able to oxidize this substrate. The K m values for the ‘B form’ differed considerably: 4 μM for β-phenylethylamine, 102 μM for tyramine, and 2.5 mM for serotonin.

Effect of lipid-depletion on the different forms of monoamine oxidase in rat liver mitochondria

In order to study the possible role of phospholipids in the genesis of different forms (‘A’ and ‘B’) of monoamine oxidase (MAO), rat liver mitochondrial monoamine oxidase was compared in mitochondria before and after lipid-depletion by extraction with aqueous methyl ethyl ketone with respect to substrate specificity and inhibitor sensitivity. With serotonin (substrate for the ‘A form’ of the enzyme) 7 per cent of the activity in the mitochondrial preparation was recovered after extraction, while 80 per cent was recovered with β-phenylethylamine (substrate for the ‘B form’ of the enzyme) and 3 per cent with tyramine (which is supposed to be a substrate for both forms). A comparison of the sensitivity to the inhibitors clorgyline (‘A form’ inhibiting) and deprenil (‘B form’ inhibiting) before and after extraction also showed that the ‘B form’ of the enzyme was almost exclusively recovered in the lipid-depleted residue. From extraction experiments performed on mitochondria with either the ‘A’ or the ‘B form’ of the enzyme selectively inhibited with clorgyline or deprenil, respectively, it could be concluded that no transformation of the ‘A form’ into ‘B form’ occurred as a result of the extraction. After extraction of mitochondria in which both forms of monoamine oxidase had been labelled with the irreversible inhibitor [ 14C]pargyline most of the radioactivity was found in the lipidzdepleted residue. This indicates that the ‘A form’ was not liberated from the membranes by the extraction, but was still present in the membrane residues in an inactivated state. The results do not support the hypothesis that the multiple functional forms of monoamine oxidase are explained by the binding of different amounts of membrane material to one single enzyme species.