Rat Liver Mitochondrial Monoamine Research Articles

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.

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 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.

Species Differences in the Selective Inhibition of Monoamine Oxidase (1-methyl-2-phenylethyl)hydrazine and its Potentiation by Cyanide

The potentiating effects of cyanide on the inhibition of rat liver mitochondrial monoamine oxidase-A & B and of ox liver mitochondrial MAO-B by pheniprazine [(1-methyl-2-phenylethyl)hydrazine] has been studied. Pheniprazine was shown to behave as a mechanism-based MAO inhibitor. For rat liver MAO-B, the initial non-covalent step was characterized by dissociation constant (K (i)) of 2450 nM and the first-order rate constant (k (+2)) for the covalent adduct formation was 0.16 min(-1). As a reversible inhibitor it was selective towards rat liver MAO-A (K (i) = 420 nM) but the rate of irreversible inhibition of that enzyme was considerably slower (k (+2) = 0.06 min(-1)). MAO-B from ox liver more closely resembled MAO-A from the rat in sensitivity to reversible inhibition by pheniprazine (K (i) = 450 nm) but it was closer to rat liver MAO-B in rate of irreversible inhibition (k (+2) = 0.29 min(-1)). The K (i) values were significantly decreased in the presence of KCN but there was little effect on the k (+2) values. However, sensitivities of the different enzymes to KCN varied widely and considerably higher concentrations of KCN were required for this effect to be apparent with the rat liver mitochondrial MAO-A than with MAO-B from rat and ox liver. The kinetic behaviour of cyanide activation was consistent with partial (non-essential) competitive activation in all cases.

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.

Nonselective inhibition of monoamine oxidases A and B by activators of soluble guanylate cyclase.

Several activators of soluble guanylate cyclase were investigated as potential inhibitors of rat liver mitochondrial monoamine oxidases (MAO) A and B. They all fitted into the previously designed "molds" of substrate-inhibitor binding sites of these enzymes. However, only two of them, NO donors (7-nitro-benzotetrazine-1,3-dioxide (7-NBTDO) and benzodifuroxan), caused nonselective inhibition of MAO A and MAO B with IC(50) values of 1.3-1.6 and 6.8-6.3 microM, respectively. The inhibitory effect on both MAO A and MAO B was reduced by mitochondria wash suggesting reversible mode of the enzyme inhibition. There was no correlation between potency of MAO inhibition and activation of human platelet soluble guanylate cyclase. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) had no effect on the manifestation of MAO inhibition by benzodifuroxan and 7-NBTDO; however, at 50 microM concentration carboxy-PTIO caused potent inhibition of MAO A with minor effect on MAO B activity. The data suggest that nonselective inhibition of MAO A and MAO B by benzodifuroxan and 7-NBTDO can be attributed to the properties of the chemical structures of these compounds. The results of the present study demonstrate a real possibility for the development of a new generation of effective reversible nonselective MAO inhibitors exhibiting equal inhibitory activity with respect to both MAO A and MAO B.

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.

The interactions of milacemide with monoamine oxidase

The interactions of the anticonvulsant drug milacemide (2- n-pentylaminoacetamide) with rat liver mitochondrial monoamine oxidases-A and -B have been studied. The compound acts as a substrate for the B-fonn of the enzyme, with an apparent K m value of 49 ± 4.7 μM and a V max value of 1.1 ± 0.2 nmol/min/mg. It is also a time-dependent irreversible inhibitor of that enzyme. Any activity of monoamine oxidase-A towards this substrate was too low to allow accurate determinations to be made by either luminometric determination of H 2O 2 formation or spectrophotometric coupling of aldehyde formation to NAD + reduction in the presence of aldehyde dehydrogenase. Milacemide was a reversible competitive inhibitor towards monoamine oxidase-A. The inhibitor constant ( K i was 115 ± 35 μM indicating a higher affinity than that towards monoamine oxidase-B, which was also competitively inhibited in the absence of enzyme-inhibitor preincubation ( K i = 331 ± 185 μM). Determination of the formation of H 2O 2 and the aldehyde product of the oxidative cleavage of milacemide by purified monoamine oxidase-B from ox liver indicated that cleavage resulted solely in the formation of pentanal and glycinamide. There was no evidence for alternative cleavage to pentylamine and oxamaldehyde.

Lipid peroxidation affects catalytic properties of rat liver mitochondrial monoamine oxidases and their sensitivity to proteolysis

1.1. Lipid peroxidation (LPO) in rat liver mitochondria decreased the activity of monoamine oxidase (MAO) with physiological substrates serotonin and 2-phenylethylamine (by 15–30%) and induced deamination of glucosamine, which was highly sensitive to selective MAO A inhibitor pirlindole.2.2. The LPO-induced changes in catalytic properties of MAOs are accompanied by their increased susceptibility to trypsinolysis, however sensitivity to inhibition by imipramine, chlorpromazine and spermine are insignificantly changed.3.3. It is suggested that these results reflect LPO-induced conformational changes of enzyme molecules in membrane rather than their membrane topography.

The inhibition of monoamine oxidase by brofaromine

The inhibition of rat liver mitochondrial monoamine oxidase-A (MAO-A) by brofaromine was time-dependent at low enzyme and inhibitor concentrations. The apparent sensitivity to inhibition decreased when the concentration of the mitochondrial preparation was increased. After preincubation of the enzyme with brofaromine repeated washing of the preparation, by sedimentation and resuspension, resulted in a gradual recovery of activity. This occurred more slowly than was the case when the reversible inhibitor amphetamine was used. After incubation with radioactively-labeled brofaromine the loss of radioactivity also occurred slowly. After incubation with radioactively-labeled pargyline polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphage (SDS-PAGE) showed the radioactivity to be associated with a peptide of approximate M r 50,000, corresponding to the subunit of MAO. Pretreatment with unlabeled pargyline depressed this labeling by pargyline, indicating the latter compound to bind to the active-site of the enzyme. Labeling experiments with radioactive brofaromine indicated that there was a high degree of non-specific binding but that no significant radioactivity remained associated with the enzyme on SDS-PAGE. Chromatographie techniques and determination of H 2O 2 liberation indicated that, in liver there was no appreciable metabolism of brofaromine under the conditions used in the inhibition experiments. These data indicate brofaromine to be a tight-binding, but reversible inhibitor of MAO.

Deuterium isotope effect of phenelzine on the inhibition of rat liver mitochondrial mono amine oxidase activity

Phenelzine is a suicide monoamine oxidase (MAO) inhibitor with antidepressant properties. The present study compares the inhibition of rat liver mitochondrial MAO by phenelzine and 1,1dideuterated phenelzine and the metabolism of these drugs by that enzyme. Phenylacetaldehyde, which was measured by a high performance liquid Chromatographic procedure, was found to be the major metabolite of phenelzine after incubation with MAO. The time-courses of aldehyde formation were non-linear due to the time-dependent inhibition of MAO. The reaction rate was reduced substantially when the hydrogen atom in the 1-carbon position was replaced by deuterium. The V H V D value was 3.1, indicating a primary isotope effect. Such a substitution of deuterium in the phenelzine molecule did not affect significantly the initial reversible inhibition of MAO, which was determined by comparison of their K i values. The irreversible inhibition, as estimated from ic 50 values, however, was potentiated substantially by deuteration. These results support the notion that the irreversible inhibition of MAO activity by phenelzine proceeds via a phenylethyldiazene intermediate, which reacts with the enzyme to form a covalent adduct. An alternative pathway involving hydrogen abstraction from carbon-1 of phenelzine or via rearrangement of the diazine on the enzyme surface could occur to form a phenylethylidene hydrazine intermediate which would subsequently be hydrolyzed to phenylacetaldehyde. The reduction in the rate of phenylethylidene hydrazine formation due to the isotope effect could lead to the accumulation of phenylethyldiazene intermediate and thus potentiate the inhibition of MAO activity.

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.

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 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.