Molecular weight of mitochondrial type B MAO in various organs of guinea pig

Molecular weights of mitochondrial type B monoamine oxidase (MAO) in guinea pig brain, liver and kidney were estimated, and their identities and multiplicity were studied. We ascertained what concentration of 3H-pargyline bound to type B MAO specifically from the inhibition curve toward serotonin (5-HT) and beta-phenylethylamine (beta-PEA) by pargyline. Pargyline irreversibly binds to FAD in MAO at a one to one molecular ratio. 3H-pargyline bound to type B MAO specifically and irreversibly by incubation for 5 hr at 37 degrees C, and SDS-disc electrophoresis was carried out using 3H-pargyline as a tracer. The molecular weight of MAO was estimated after specific binding of pargyline was corrected for non-specific binding. The molecular weight of type B MAO in every organ was found to be 60,000, giving a single peak after solubilization with 6% SDS, but several peaks at higher molecular weight were found in each organ after solubilization with 2% SDS. In the brain, there appeared to be a peak of 100,000, and it was suggested that the MAO existed as a dimer which was composed of a FAD containing subunit and a low molecular weight subunit containing no FAD. In the liver, there appeared to be peaks of 120,000 and 240,000, and it was suggested that the MAO existed as a dimer and tetramer. In the kidney, there appeared to be a peak of 180,000, and MAO was suggested to exist as a trimer.     

Chronic MAO A and MAO B inhibition decreases the 5-HT1A receptor-mediated inhibition of forskolin-stimulated adenylate cyclase

The effect of chronic administration of various monoamine oxidase (MAO) inhibitors on the ability of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) to inhibit forskolin-stimulated adenylate cyclase activity was studied. Groups of 12 rats were given either saline, (E)-beta-fluoromethylene-m-tyrosine (MDL 72394 0.25 mg/kg p.o.), clorgyline (1 mg/kg p.o.), selegiline (1 mg/kg p.o.) or tranylcypromine (5 mg/kg p.o.) once a day for 21 days. Biochemical determinations were made 72 h after the final dose. MDL 72394 and tranylcypromine produced a nonselective inhibition of MAO but clorgyline and selegiline selectively inhibited MAO A and MAO B respectively. All treatments that inhibited MAO A also increased tissue levels of 5-HT. Chronic treatment with MDL 72394, clorgyline or tranylcypromine reduced the ability of 8-OH-DPAT to inhibit forskolin-stimulated adenylate cyclase activity. These data suggest that chronic nonselective and chronic MAO A inhibition causes a down-regulation of the 5-HT1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity.     

Role of MAO A and B in neurotransmitter metabolism and behavior.

MAO (monoamine oxidase) A and B are key isoenzymes that degrade biogenic and dietary amines. MAO A preferentially oxidizes serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE), whereas MAO B preferentially oxidizes phenylethylamine (PEA). Both forms can oxidize dopamine (DA). However, the substrate specificity overlap and the in vivo function of these two isoenzymes is not clear. Recently, we have shown that MAO A and B knock-out (KO) mice exhibit distinct differences in neurotransmitter metabolism and behavior. MAO A KO mice have elevated brain levels of 5-HT, NE and DA and manifest aggressive behavior similar to men with a deletion of MAO A. In contrast, MAO B KO mice do not exhibit aggression and only levels of PEA are increased. Both MAO A and B KO mice show increased reactivity to stress. Taken together, these results suggest that MAO A and B have distinctly different roles in monoamine metabolism. Further, these mice are valuable models for investigating the role of monoamines in psychoses and neurodegenerative and stress-related disorders.     

Influence of MAO A and MAO B on the inactivation of noradrenaline in the saphenous vein of the dog

Specific inhibitors of MAO A (clorgyline 0.1 mumol/l) and of MAO B [(-)deprenyl 10 mumol/l] were used in dog saphenous vein strips in order to study the relative influence of the two types of MAO on: 1. Termination of contractile response to exogenous noradrenaline (NA); 2. Metabolism and accumulation of exogenous (3H)-NA; 3. Metabolism of 3H-NA released by electrical stimulation. To study the termination of contractile response to exogenous NA, the oil immersion technique was used to determine the time for half-relaxation. The experiments were performed on strips with or without treatment with cocaine 10 mumol/l (to inhibit neuronal uptake) plus U-0521 100 mumol/l (to inhibit catechol-O-methyl transferase) before and after exposure to MAO inhibitors. Clorgyline, but not (-)deprenyl enhanced significantly the time for half-relaxation of the strips, whether cocaine was present or not. To study the metabolism and accumulation of exogenous 3H-NA, the strips were incubated (with or without preincubation with cocaine) with 3H-NA 0.23 mumol/l and 2.3 mumol/l in the presence and in the absence of MAO inhibitors. The formation of deaminated metabolites was significantly reduced by clorgyline, but not by deprenyl. To study the metabolism of 3H-NA released by electrical stimulation, the strips were incubated with 3H-NA 1.4 mumol/l. In the presence of cocaine and U-0521, field stimulation was applied during two periods of 5 min (10 Hz, 100 V, 2 ms), in the absence or presence of MAO inhibitors. Under these experimental conditions clorgyline, but not deprenyl, abolished DOPEG formation, without affecting the other metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irreversible Inhibition of Rat Liver Mitochondrial MAO A and MAO B by Enantiomers of Deprenyl and α-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 α-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.     

Molecular basis of chronopharmaceutics

Many pathophysiological circumstances vary during 24 h periods. Many physiologic processes undergo biological rhythms, including the sleep-wake rhythm and metabolism. Disruptive effect in the 24 h variations can manifest as the emergence or exacerbation of pathological conditions. So, chronotherapeutics is gaining increasing interest in experimental biology, medicine, pharmacy, and drug delivery. This science and the plethora of information should be used intelligently for optimizing the effectiveness and safety of the drug, relying on the timing of drug intake. These chronopharmacological findings are affected by not only the pharmacodynamics but also pharmacokinetics of drugs. The mammalian circadian pacemaker is located in the suprachiasmatic nucleus. The molecular mechanisms are associated with Clock genes that control the circadian rhythms in physiology, pathology, and behavior. Clock controls several diseases such as metabolic syndrome, cancer, and so on. CLOCK mutation influences the expression of both rhythmic and nonrhythmic genes in wild-type tissues. These genotypic changes lead to phenotypic changes, affecting the drug pharmacokinetic and pharmacodynamic parameters. This review is intended to elaborate system regulating biological rhythms and the applicability in pharmaceutics from viewpoints of the intraindividual and interindividual variabilities of Clock genes.     

Molecular basis of structure-activity relationships between salphen metal complexes and human telomeric DNA quadruplexes

The first X-ray crystal structures of nickel(II) and copper(II) salphen metal complexes bound to a quadruplex DNA are presented. Two structures have been determined and show that these salphen-metal complexes bind to human telomeric quadruplexes by end-stacking, with the metal in each case almost in line with the potassium ion channel. Quadruplex and duplex DNA binding is presented for these two and other related salphen complexes, all with side-chains terminating in pyrrolidino end-groups and differing patterns of substitution on the salphen core. The crystal structures are able to provide rationalizations for the structure-activity data, and in particular for the superior quadruplex-binding of the nickel complexes compared to that of the copper-containing ones. The complexes show significant antiproliferative activity for the compounds in a panel of cancer cell lines. They also show telomerase inhibitory activity in the telomerase TRAP-LIG assay.     

Molecular basis of insulin action

Insulin is the main anabolic and anticatabolic hormone in mammals. The stimulatory effect of insulin on glucose uptake in muscle and adipose tissue is a consequence of the rapid translocation of GLUT4 glucose transporters from an intracellular site to the cell surface. The actions of insulin are initiated by hormone binding to its cell surface receptors. Insulin receptors are ligand-stimulated protein tyrosine kinases and phosphorylate a number of proteins, known as insulin receptor substrate proteins. Insulin resistance has been recognized as a main pathogenic factor in the development of type 2 diabetes, and has been associated with dyslipidemia, hypertension, endothelial dysfunction, inflammation and coagulative state. The current challenge is the study of impaired insulin signaling pathways leading to beta-cell dysfunction and its progression to type 2 diabetes, as well as control of chronic inflammation processes that may improve insulin action.     

Molecular properties of monoamine oxidases A and B

Monoamine oxidases A and B (MAO-A and B) catalyze the oxidative catabolism of biogenic amines and xenobiotics. Investigation of these mitochondrial membrane proteins shows that they differ in substrate preference, inhibitor specificity, tissue and neuronal cell distribution, immunological properties, and nucleotide and deduced amino acid sequences. Comparisons of MAO-A and B from the human, bovine, and rat species show strikingly high similarity (85–88%) in the amino acid sequences of each enzyme. Furthermore, three regions in MAO-A and B have sequence identities across species of 78, 88, and 86%. These regions correspond to a nucleotide-binding site near the N-terminal end that is found in the vast majority of enzymes that require flavin adenine dinucleotide (FAD), a region of unknown function, and the FAD-binding site toward the C-terminal end. Genomic clones of MAO-B which span almost the entire gene (>40 kb) have been isolated, restriction mapped, and partially sequenced. Likewise, genomic clones of MAO-A that correspond to the 3-flanking region have also been investigated. Current studies which focus on identification of the promotor and regulatory sequences should help to establish why MAO-A and B are localized in different subsets of neurons in brain.     

Serotonin oxidation by type B MAO of rat brain

The two MAO types in rat brain can be selectively inhibited by administering intraperitoneal injections of clorgyline or pargyline in suitable doses. Brain mitochondria prepared from such animals exhibit type B or type A MAO activity, respectively. In vitro clorgyline and deprenyl dose-response curves confirmed the purity of the enzyme preparations. Specific activities and K_m values of such preparations were determined for tyramine, serotonin and benzylamine. Type B and type A MAO were found to oxidize serotonin and benzylamine. respectively, although they had low affinities. Serotonin oxidation by mitochondria prepared from clorgyline treated animals showed type B characteristics also in its heat inactivation time course.     

Molecular basis of insulin receptor function

Insulin regulates a wide spectrum of metabolic processes in a variety of tissues. In addition, insulin is a potent growth factor, promoting normal cell growth and division in cultured cells, though the physiological importance of such actions in man is less certain. Some of the effects of insulin are very rapid and apparent within seconds, for example those on transport processes and the activity of regulatory enzymes. Other effects, such as those on enzyme concentrations and cell growth, may be manifest only over a period of many minutes or even hours. Such differences in time dependence may be more a reflection of the nature of the processes themselves than an indication of different signalling mechanisms. However, it is still unclear whether the regulation of diverse metabolic processes by insulin involves just one or multiple distinct primary intracellular signals. We will review here what is at present known about the structure and activity of the receptor, and current ideas on signalling mechanisms.     

Molecular basis of alpha-KTx specificity.

Potassium channel inhibitor peptides from scorpion venom, alpha-KTx, have greatly advanced our understanding of potassium channel structure and function, Because of their high affinity interaction with the outer pore, alpha-KTx's have aided, in identification of amino acids lining the pore and of proteins constituting functional channels. The alpha-KTx's display a large range of affinities for different potassium channels with differences in binding free energy exceeding approximately 8 kcal/mol. These differences in affinities are the foundation of alpha-KTx specificity and have aided in revealing the physiological and patho-physiological roles of potassium channels. The alpha-KTx subfamilies 1-3, display gross differences in specificity for maxi-K vs. KV channels. However, many potassium channels are largely untouched by alpha-KTx's. Differences in toxin binding free energy provide a quantitative framework for defining specificity. As a practical criterion for specificity a minimum binding free energy difference of 2.72 kcal/mol is proposed. Binding free energy differences for wild-type and mutant toxins and channels can point to amino acids underlying specificity and to unique features of potassium channel outer pores. Known 3D structures of potassium channels in combination with CLUSTALW sequence alignment of over 60 potassium channels reveal significant variation in alpha-KTx binding domains. Structure-based homology models of potassium channels complexed with alpha-KTxs, in combination with measurements of toxin binding free energy, will further our understanding of the molecular basis of alpha-KTx specificity.     

Tele-methylhistamine is a specific MAO B substrate in man

Tele-methylhistamine, the first metabolite of histamine in tissues which lack diamine oxidase, is shown to be a substrate for human MAO B. Human liver homogenates were incubated with ³H-tele-methylhistamine and the products separated using thinlayer chromatography. The major product was 3-methylimidazoleacetic acid, the oxidatively deaminated metabolite of tele-methylhistamine. The reaction was inhibited by low concentrations of (-)deprenyl, the specific MAO B inhibitor. Tele-methylhistamine was also found to inhibit competitively the oxidation of phenylethylamine, but not that of 5-hydroxytryptamine, providing further evidence that it is oxidized by MAO B itself and not a related enzyme. This finding implies that (-)deprenyl and other MAO inhibitors used clinically may interfere with histamine metabolism.     

Molecular basis for the interaction of four different classes of substrates and inhibitors with human aromatase

Aromatase cytochrome P450 (CYP19) converts androgen to estrogen. In this study, the interactions of four classes of compounds, 17beta-estradiol (the product of aromatase), 17-methyltestosterone (a synthetic androgen), dibenzylfluorescein (a synthetic substrate of aromatase), and coumestrol (a phytoestrogen), with aromatase were investigated through spectral analysis using purified human recombinant aromatase and site-directed mutagenesis studies using CHO cells expressing wild-type human aromatase or five aromatase mutants, E302D, D309A, T310S, S478T and H480Q. Spectral analysis showed that a type I binding spectrum was produced by the binding of 17-methyltestosterone to aromatase and a novel binding spectrum of aromatase was induced by dibenzylfluorescein. Mutagenesis experiments demonstrated that residues S478 and H480 in the beta-4 sheet play an important role in the binding of all four compounds. Computer-assisted docking of these compounds into the three-dimensional model of aromatase revealed that: (1) weak interaction between 17beta-estradiol and the beta-4 sheet of aromatase facilitates the release of 17beta-estradiol from the active site of aromatase; (2) 17-methyl group of 17-methyltestosterone affects its binding to aromatase; (3) dibenzylfluorescein binds to the active site of aromatase with its O-dealkylation site near the heme iron and residue T310; and (4) coumestrol binds to aromatase in a manner such that rings A and C of coumestrol mimic rings A and B of steroid. These structure-function studies help us to evaluate the structural model of aromatase, and to accelerate the structure-based design for new aromatase inhibitors.