In vitro inhibition of human platelet monoamine oxidase by phenothiazine derivatives

Nineteen phenothiazines were tested for in vitro inhibition of human platelet type B monoamine oxidase (MAO). The inhibition potency was highly dependent on structures of their side chains. The inhibition was most potent for drugs with (hydroxyethyl-piperazinyl)propyl chains followed in decreasing order by those with (N-methylpiperazinyl)propyl, (2-dimethylamino-2-methyl)ethyl and 3-dimethylaminopropyl chains. Kinetic analyses were carried out for promazine, promethazine, perazine and perphenazine as representatives of each group; the four drugs showed competitive inhibition, and Ki values of 124, 31.4, 19.2 and 22.6 microM, respectively.     

Purification of human blood platelet monoamine oxidase

Monoamine oxidase B has been purified from human blood platelets 185-fold to a specific activity of 113 nmole/min/mg protein by a combination of Triton X-100 solubilization and ion exchange chromatography. A protein fraction corresponding to 58,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identified as monoamine oxidase by its ability to bind [3H]Pargyline.     

Effects of tricyclic antidepressants upon human platelet monoamine oxidase

Tricyclic antidepressant drugs were found to inhibit human platelet MAO. The I50 for the inhibition by amitriptyline was 4 × 10^?6 M, 1.6 × 10^?5 M, and 2 × 10^?4 M when phenylethylamine, tryptamine, and benzylamine were used as substrates. Amitriptyline exhibited noncompetitive inhibition with the substrates phenylethylamine and tryptamine but competitive inhibition with benzylamine. Solubilization and partial purification of platelet MAO did not alter the inhibitory effects of tricyclics. Treatment of the partially purified enzyme with the chaotropic agent sodium perchlorate produced only a slight increase in the inhibition constant for amitriptyline. Our findings suggest that selective inhibition of phenylethylamine oxidation may mediate the antidepressant actions of tricyclics. In addition, our studies provide some evidence for the existence of multiple catalytic sites of MAO activity in the human platelet.     

Measurement of platelet monoamine oxidase activity in healthy human volunteers

Platelet MAO activity of 176 healthy volunteers was studied. Activity of the enzyme was found intraindividually stable, repeated measurements in the same individuals performed 3 weeks and one year later revealed only minimum changes. Platelet MAO activity was found to be significantly higher in females than in males. No age dependence of the measured values could be demonstrated. Enzyme activity values determined with different substrates showed a significant positive correlation. A significant activation of the enzyme was found after the addition of platelet poor plasma to the platelet preparation.     

Activation of platelet monoamine oxidase by plasma in the human.

A pronounced activation of platelet monoamine oxidase (MAO) by human plasma has been observed. The activation was substrate selective, since serotonin, $\text{p}$-tyramine, dopamine and benzylamine were much more effective than β-phenylethylamine or tryptamine. The activator(s) in the plasma was heat stable but labile to acid hydrolysis and treatment with lipase and protease. The plasma was also found to be capable of activating partially purified MAO obtained from rat liver mitochondria. Phospholipids such as phosphatidylethanolamine were shown to activate MAO.     

Liver monoamine oxidase activity of the lamprey Lampetra fluviatilis. The substrate-inhibitor specificity

Based on the data of substrate-inhibitor analysis with the use of specific inhibitors-deprenyl, chlorgilin, and specific substrates-serotonin, noradrenalin, benzylamine, β-phenylethylamine and N-methylhistamine, one molecular form of monoamine oxidase (MAO) was suggested to possibly exist in liver of mature individuals of the European lamprey Lampetra fluviatilis. The kinetic parameters of monoamine oxidase deamination of eight substrates were determined, which indicates the large spectrum of substrate specificity of MAO from the lamprey liver. The studied enzyme does not deaminate histamine and putrescine and is not sensitive to 10^?2 M semicarbaside. Results of the study of the substrate-inhibitor specificity allow us to suggest some resemblance in catalytic properties of the lamprey liver MAO and the mammalian MAO of the form A. The low activity of the enzyme revealed at deamination of all used substrates seems to be associated with low detoxifying function of the lamprey liver.     

Heterogeneity of platelet monoamine oxidase in man

Multiplicity of platelet MAO was studied. Multiple forms of enzyme was separated with the use of 1.5% Tritone x-100 and, 1.3 M urea at pH 7.4 in, 0.01 M K-Na phosphate buffer. Three forms of MAO--MAO-I, MAO-II, and MAO-III were obtained under separation of solubilized proteins by affinity chromatography on AN-Sepharose 4B. Deprenyl in 10(-5) M inhibited all forms of the activity completely. Relation between multiple forms of the brain and platelet was discussed.     

Increased human platelet monoamine oxidase activity during lithium carbonate therapy

Human platelet monoamine oxidase (MAO) activity was assessed, using benzylamine C¹⁴ as the substrate, in ten patients who received a trial of lithium carbonate treatment. In all ten patients there was an increase of the median MAO activity during the lithium treatment period in comparison to the median MAO activity during a placebo treatment period. This increase in platelet MAO activity is consistent with reports by other investigators that lithium treatment is associated with increased oxidative deamination of monoamines in both animals and humans.     

Type B monoamine oxidase activity in human brain malignant tumors

An increase of monoamine oxidase (MAO) activity was observed in Central Nervous System (CNS) malignant tumors, but the isoform responsible was not identify (Marcozzi et al., 1985). In the present work we report additional data in order to ascertain whether the type A or B MAO isoform is increased in some malignant human tumors of CNS. In the homogenated tissues the amine oxidase activity was determined by the chemiluminescent method, using different and specific substrates or inhibitors of MAO A and B and copper-dependent enzymes. 19 samples from 4 different types of tumors and relative peritumoral tissues were analysed. The highest activity of was imputable to type B MAO.     

Partial amino acid sequence analysis of human placenta monoamine oxidase A and bovine liver monoamine oxidase B

We have prepared peptide maps from human placenta monoamine oxidase type A (MAO-A) and bovine monoamine oxidase type B (MAO-B) and determined the amino acid sequences of 21 of these peptides. These sequences have been compared to the cDNA deduced amino acid sequences of human MAO-A and -B. A result of special interest is the identification of two sets of MAO-A peptides which have sequences different from those deduced from cDNA sequences. This observation is consistent with the notion that MAO-A may be composed of at two subunits which are similar but not identical in primary amino acid sequence.     

Low platelet monoamine oxidase activity in cigarette smokers

The activity of platelet monoamine oxidase was found to be significantly lower in normal female subjects who smoked at least 5 cigarettes per day than in non-smokers. The platelet MAO activity of individuals who had given up smoking was not significantly different from the activity for non-smokers. The difference in activities between smokers and non-smokers, due entirely to a V_max rather than a K_m change, was not due to a direct effect of nicotine upon the platelet MAO. In addition, platelet-poor plasma from smokers activated platelet MAO in an identical manner to that from non-smokers. The significance of these results are discussed in terms of personality characteristics such as impulsivity and sensation seeking, that may be related to both smoking and to low MAO activity.     

The in vitro insertion of monoamine oxidase B into mitochondrial outer membranes

Bovine monoamine oxidase (MAO) B has been synthesized in vitro using a reticulocyte lysate translation system directed by bovine liver poly(A)+ RNA. The newly synthesized enzyme apparently lacks a cleavable N-terminal extension, but MAO B is readily incorporated into mitochondria or isolated mitochondrial outer membranes prepared from rat liver. ATP is not required for the binding of the newly synthesized enzyme to the outer membranes, but is necessary for the insertion of MAO B into these membrane vesicles. The ATP is not required to generate a mitochondrial membrane potential as assembly occurs under conditions that preclude either the formation or the maintenance of the potential. MAO B will bind to but not become incorporated into outer membrane vesicles which have been treated with trypsin, suggesting that the insertion of MAO B also depends on protein factors present on the outer membranes.     

High-level expression of human liver monoamine oxidase B in Pichia pastoris

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.     

Kinetic analysis of platelet monoamine oxidase in chronic schizophrenia

Km and Vmax values for platelet monoamine oxidase (MAO) were determined in 16 chronic schizophrenics and 18 controls utilizing three substrates, tyramine (TYR), benzylamine (BZ), and phenylethylamine (PEA). In the chronic schizophrenics decreased Km and Vmax values were found for TYR and BZ but not PEA. When prior neuroleptic drug exposure was considered, a trend toward lower kinetic parameters was found in schizophrenics with a history of prior neuroleptic usage. We conclude that platelet MAO activity is, in chronic schizophrenics, both quantitatively reduced and qualitatively different from control enzyme. We suggest that the measurement of Km in addition to the measurement of Vmax may be a useful biological marker for chronic schizophrenia providing that the appropriate substrates are employed.     

Comparative substrate-inhibitor analysis of mink liver monoamine oxidases

Comparative substrate-inhibitor analysis of catalytic properties of liver monoamine oxidases (MAO) was performed in the mature males of the American mink Mustela vison and the European mink Mustela lutreola. The action on the MAO activity was studied of alkaloids of the benzo[c]phenanthridine group: sanguinarine and chelidonine, diisoquinoline alkaloid berberine, medicinal agents “Ukrain” and “Sanguirythrin” as well as derivatives of 2-propylamine: deprenyl and chlorgylin. The latter turned out to be irreversible inhibitor of the MAO A form, whereas deprenyl-irreversible inhibitor of the MAO B form in both studied mink species. The selectivity of action of each inhibitor on the corresponding liver MAO form for the species M. vison was one order of magnitude stronger than for the species M. lutreola. All studied alkaloids as well medicinal agents on their basis have been shown to be specific irreversible inhibitors of the intermediate strength of the liver MAO A form of both mink species. They inhibit the enzymatic deamination of serotonin, tyramine, and tryptamine without affecting the deamination reaction of benzylamine and β-phenylethylamine (at concentrations of 10 mM and lower). Out of five studied isoquinoline agents, the medication “Ukrain” and alkaloid chelidonine have the highest inhibitory action; the agent “Sanguirythrin” and alkaloids berberine and sanguinarine produce the weaker monoamine oxidase effect. The revealed specificity of action of the studied inhibitors is an indirect evidence for the presence in the liver enzymes of both mink species, like in the rat liver enzyme, of several molecular forms.     

Comparative substrate-inhibitor analysis of liver monoamine oxidases of minks

Comparative substrate-inhibitor analysis of catalytic properties of liver monoamine oxidases (MAO) was performed in the mature males of the American mink Mustela vison and the European mink Mustela lutreola. The action on the MAO activity was studied of alkaloids of the benzo[c]phenanthridine group: sanguinarine and chelidonine, diisoquinoline alkaloid berberine, medication agents Ukrain and Sanguirythrin as well as derivatives of 2-propylamine: deprenyl and clorgylin. The latter turned out to be irreversible inhibitor of the MAO A form, whereas deprehyl--irreversible inhibitor of the MAO B form in both studied mink species. The selectivity of action of each inhibitor on the corresponding liver MAO form for the species M. vison was one order of magnitude stronger than for the species M. lutreola. All studied alkaloids as well medication agents on their basis have been shown to be specific irreversible inhibitors of the intermediate strength of the liver MAO A form of both mink species. They inhibit the enzymatic deamination of serotonin, tyramine, and tryptamine without affecting the deamination reaction of benzylamine and beta-phenylethylamine (at concentrations of 10 mM and lower). Out of the studied five isoquinoline agents, the medication Ukrain and alkaloid chelidonine have the highest inhibitory action; the agent Sanguirythrin and alkaloids berberine and sanguinarine produce the weaker monoamine oxidase effect. The revealed specificity of action of the studied inhibitors is an indirect evidence for the presence in the liver enzymes of both mink species, like in the rat liver enzyme, of several molecular forms.