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 6.3-6.8 M, 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 M 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.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: ¹⁴C-labeled tryptamine, dopamine and kynuramine. With dopamin as substrate, the enzyme activity showed decline during 24 and 48 h starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decreased after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Studies of monoamine oxidases: properties of the enzyme in bovine and rabbit brain mitochondria

Brain mitochondria were prepared from rabbit and bovine cerebral cortex and the purity and intactness of the preparation assessed through the use of enzyme markers and electron microscopy. Enzymatic properties of monoamine oxidase were studied in the purified mitochondrial preparations which were essentially devoid of major contamination by other organelles, especially microsomes. Five substrates were used for characterization of the enzyme: dopamine, kynuramine, serotonin, tryptamine and tyramine. It was found that there was considerable substrate variation in the properties, but in general, the two species showed similar characteristics. The more pertinent findings were: (1) apparent K_m values ranged from 1.1 ± 10^?5m for tryptamine to 2.5 ± 10^?4m for dopamine; (2) substrate specificity from V_max values in decreasing order was tyramine > dopamine > kynuramine > serotonin > tryptamine for the bovine enzyme and tyramine > kynuramine > dopamine > serotonin > tryptamine for rabbit; (3) there appeared to be three distinct pH optima according to substrate: pH 7.5 for phenylethylamines, pH 8.2–8.5 for the indolylamines and pH 9.1 for kynuramine; and (4) the activity with tyramine was highly sensitive to increased oxygen tension while kynuramine showed no sensitivity. It is proposed that the properties of monoamine oxidase, a membrane-bound enzyme, might be influenced by the microenvironment and results are also discussed in terms of multiple forms or multiple activity sites on a single form.     

Purification and Molecular Analysis of a Monoamine Oxidase Isolated from Narcissus tazetta

Semicarbazide-sensitive amine oxidase activity was detected in Narcissus tazetta. The enzyme was purified to homogeneity by the criterion of native polyacrylamide gel electrophoresis (PAGE) with DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose columns. The molecular mass of the enzyme, determined using a GS-520 HQ column, was estimated to be 135 kDa. SDS–PAGE yielded two bands of, 75 kDa and 65 kDa. The enzyme, which had catalytic activity for some aliphatic and aromatic monoamines, belongs to a class of monoamine oxidases (MAOs). The K _m value for n-propylamine was 5.9 × 10^?5 M. A substrate analog, 2-bromoethylamine, inhibited enzyme activity. Redox-cycling staining detected a quinone in the MAO protein. By inductively coupled plasma mass analysis, it was determined that there were 2.44 moles of copper atoms per mole of the enzyme. Protein sequence analysis revealed that there was no identity between two N-terminal residues of the 75 kDa and 65 kDa proteins of narcissus MAO.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats.

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: 14C-labeled tryptamine, dopamine and kynuramine. With dopamine as substrate, the enzyme activity showed decline during 24 and 48 h of starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decrease after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Relation of the extra-sequence of the precursor form of chicken liver δ-aminolevulinate synthase to its quaternary structure and catalytic properties

Precursor and mature forms of δ-aminolevulinate (ALA) synthase were purified to near homogeneity from chicken liver mitochondria and cytosol, respectively, and their properties were compared. The enzyme purified from mitochondria had apparently the same subunit molecular weight (65,000) as that of the native mitochondrial enzyme. The enzyme purified from the cytosol fraction, however, showed a subunit molecular weight of about 71,000, which was somewhat smaller than that estimated for the native cytosolic enzyme (73,000). The enzyme purified from liver cytosol seems to have been partially degraded by some endogenous protease during the purification, but may have the major part of the signal sequence. On sucrose density gradient centrifugation, the purified mitochondrial and cytosolic ALA synthases showed an apparent molecular weight of about 140,000, indicating that both enzymes exist in a dimeric form. The ALA synthase synthesized in vitro was also shown to exist as a dimer. Apparently the extra-sequence does not interfere with the formation of dimeric form of the enzyme. The purified cytosolic ALA synthase had a specific activity comparable to that of the purified mitochondrial enzyme. Kinetic properties of the two enzymes, such as the pH optimum and the apparent K_m values for glycine and succinyl-CoA, were quite similar. The extra-sequence does not appear to affect the catalytic properties of ALA synthase. The isoelectric point of the cytosolic ALA synthase was 7.5, whereas that of the mitochondrial enzyme was 7.1. This suggests that the extra-sequence in the cytosolic enzyme may be relatively rich in basic amino acids.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Cytochrome c oxidase in prokaryotes

Abstract Several heme aa ₃-type cytochrome c oxidases, purified from the cytoplasmic membranes of bacteria, are able to catalyze the same reactions as the structurally far more complex eukaryotic enzyme, i.e., electron transport from cytochrome c to oxygen coupled to proton translocation. However, these oxidases show a very simple subunit pattern, and moreover, individual polypeptides even have homologous amino-acid sequences. This review summarizes the present data on purified bacterial cytochrome c oxidases and relates these findings to results obtained with the mitochondrial enzymes.     

Criteria for the evaluation of the functional importance of endogenous analogues of pharmacological regulators

Criteria for the evaluation of the functional importance of endogenous analogues of pharmacological drugs are proposed. These include opposite changes in their content in opposite pathophysiological or physiological states, accompanied by corresponding changes in the functional activity of enzymes sensitive to changes in their level, and the regulation of target enzymes by physiological concentrations of such endogenous compounds. The applicability of these criteria has been demonstrated using tribulin, the endogenous family of inhibitors of monoamine oxidases.     

Effect of aluminium (Al) on brain mitochondrial monoamine oxidase-A (MAO-A) activity — anin vitro kinetic study

Effect of aluminium (Al) on rat brain mitochondrial monoamine oxidase-A (MAO-A) was studiedin vitro at three different pH (4.0, 7.4 and 9.0) values. The results have shown that Al is a non-competitive inhibitor for MAO-A. The data also showed that MAO-A inhibition by Al varies with free Al³⁺ concentration and different forms of Al under different pH conditions. Al altered the maximum velocity (V_max) of MAO-A but did not affect substrate-enzyme affinity (K_m). Al formed a strong chelation with the substrate (Kynuramine) (1:1).     

On Preparative Separation of Brain Mitochondrial Monoamine Oxidases

A method was developed for solubilization from- bovine brain stem mitochondrial fraction of monoamine oxidases deminating biogenic amines. Preparative separation of the monoamine oxidases, possessing different substrate specificities, was achieved by column chromatography on a biospecific adsorbent AH-Sepharose 4 B. The enzyme preparations thus obtained did not contain any detectable by disc-electrophoresis or isoelectrofocusing in polyacrylamide gels proteins which were devoid of the monoamine oxidase activity.     

On preparative separation of brain mitochondrial monoamine oxidases.

A method was developed for solubilization from bovine brain stem mitochondrial fraction of monoamine oxidases deminating biogenic amines. Preparative separation of the monoamine oxidases, possessing different substrate specificities, was achieved by column chromatography on a biospecific adsorbent AH-Sepharose 4 B. The enzyme preparations thus obtained did not contain any detectable by disc-electrophoresis of isoelectrofocusing in polyacrylamide gels proteins which were devoid of the monoamine oxidase activity.     

Comparative study of catalytic properties of mink and rat liver monoamine oxidase

Comparative substrate-inhibitor analysis of catalytic properties of mitochondrial monoamine oxidase (MAO) of liver of the American mink Mustela vison Schreber and of liver of Wistar rat has been performed. It has been found that MAO of mink, like MAO of rat, has properties of classic mammalian MAO: it deaminates tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine and does not deaminate histamine as well as does not have sensitivity to semicarbazide. Study of kinetics of the monoamine oxidase deamination revealed both qualitative and quantitative differences between these enzymes. Specificity of action on MAO-A form of four irreversible inhibitors—acridine derivatives—has been shown; this specificity was several times higher for the mink liver MAO than for the rat liver MAO. It is suggested that the liver MAO of both species of the studied animals has several isoenzyme forms or several centers of the substrate binding.     

Molecular characteristics of tissue-bound semicarbazide-sensitive amine oxidase (SSAO) in guinea pig tissues

Various mammalian tissues contain a tissue-bound amine oxidizing enzyme distinct from mitochondrial outer membrane enzyme, monoamine oxidase (MAO, EC 1.4.3.4), termed semicarbazide-sensitive amine oxidase (SSAO, EC 1.4.3.6). An increase in SSAO activity was found in patients suffering from vascular disorders such as diabetes and diabetic complications. It has previously been shown that 2-bromoethylamine (2-BEA) is a potent, and selective suicidal inhibitor of tissue-bound SSAO. The aim of this study was to investigate the interaction of this suicidal SSAO inhibitor with the tissue-bound enzyme in guinea pig lung, kidney, stomach, and heart homogenates. The conditions necessary for this inhibitor to titrate the concentrations of this enzyme were also determined. 2-BEA appears to interact with SSAO, as reported previously for this enzyme from different sources, in a manner consistent with an irreversible, "suicide" reaction. Because of this property, 2-BEA could be used to titrate the concentrations of SSAO active centers in these tissues under the appropriate conditions employed. Although some possible non-specific binding of the inhibitor to sites other than the active center of the enzyme, metabolism of this inhibitor and/or presence of enzyme subtypes was hypothesized, the molecular characteristics of SSAO in these tissues (Km, Vmax values, enzyme efficiencies, approximate enzyme concentrations, and molecular turnover numbers) towards the substrate kynuramine (0.1 mM) at pH 7.4 and 37 degrees C have been estimated.     

In vitro andin vivo effect of chloropromazine,imipramine and lithium chloride on monoamine oxidase activity in rat brain mitochondria

Chloropromazine (CPZ) and imipramine at a concentration of 1×10^–3 M inhibit rat brain mitochondrial monoamine oxidase activity in vitro by 70 and 55% respectively, while lithium, even at a concentration of 0.05 M, inhibits the activity of this enzyme very negligibly (4%). In vivo, these drugs at a dose level of 56 mg CPZ, 76 mg Jimipramine and 76 mg lithium chloride/Kg body wt., did not cause any observable variation from normal in brain mitochondrial monoamine oxidase activity.To whom correspondence should be addressed.     

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.     

Medical implications from the crystal structure of a copper-containing amine oxidase complexed with the antidepressant drug tranylcypromine

The X-ray crystal structure of the copper-containing quinoprotein amine oxidase from E. coli has been determined in complex with the antidepressant drug tranylcypromine to 2.4 A resolution. The drug is a racemic mix of two enantiomers, but only one is seen bound to the enzyme. The other enantiomer is not acting as a substrate for the enzyme as no catalytic activity was detected when the enzyme was initially exposed to the drug. The inhibition of human copper amine oxidases could be a source of side-effects in its use as an antidepressant to inhibit the flavin-containing monoamine oxidases in the brain.     

The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities

The role of ribityl side chain hydroxyl groups of the flavin moiety in the covalent flavinylation reaction and catalytic activities of recombinant human liver monoamine oxidases (MAO) A and B have been investigated using the riboflavin analogue: N(10)-omega-hydroxypentyl-isoalloxazine. Using a rib5 disrupted strain of Saccharomyces cerevisiae which is auxotrophic for riboflavin, MAO A and MAO B were expressed separately under control of a galactose inducible GAL10/CYC1 promoter in the presence of N(10)-omega-hydroxypentyl-isoalloxazine as the only available riboflavin analogue. Analysis of mitochondrial membrane proteins shows both enzymes to be expressed at levels comparable to those cultures grown on riboflavin and to contain covalently bound flavin. Catalytic activities, as monitored by kynuramine oxidation, are equivalent to (MAO A) or 2-fold greater (MAO B) than control preparations expressed in the presence of riboflavin. Although N(10)-omega-hydroxypentyl-isoalloxazine is unable to support growth of riboflavin auxotrophic S. cerevisiae, it is converted to the FMN level by yeast cell free extracts. The FMN form of the analogue is converted to the FAD level by the yeast FAD synthetase, as shown by expression of the recombinant enzyme in Escherichia coli. These data show that the ribityl hydroxyl groups of the FAD moiety are not required for covalent flavinylation or catalytic activities of monoamine oxidases A and B. This is in contrast to the suggestion based on mutagenesis studies that an interaction between the 3'-hydroxyl group of the flavin and the beta-carbonyl of Asp(227) is required for the covalent flavinylation reaction of MAO B (Zhou et al., J. Biol. Chem. 273 (1998) 14862-14868).     

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.     

Kinetic behaviour of acid phosphatase-albumin co-polymers in homogeneous phase and under gel-immobilized conditions.

1. Antiserum raised to purified human liver monoamine oxidase was used to characterize the monoamine oxidase from human liver, brain cortex, placenta and platelets. 2. Antibodies to monoamine oxidase were purified by adsorption with a mitochondrial preparation. 3. Monoamine oxidase was present in liver particle-free supernatant as measured by enzyme activity and immunodiffusion. 4. Multiple precipitin lines were obtained on immunodiffusion analysis against the purified liver enzyme. It is proposed that this is due to either aggregation or to differential lipid binding. 5. The results suggest that the functionally different enzymes found in liver, brain cortex, platelets and placenta are immunochemically related and may be identical.