Problems with the measurement of monoamine oxidase A protein concentration in mitochondrial preparations Revised molecular activities and implications for estimating ratios of MAO A:MAO B molecules from radiochemical assay data

doi:10.1016/0006-2952(91)90595-V

Biochemical Pharmacology

Volume 42, Issue 10, 24 October 1991, Pages 1953-1959

https://doi.org/10.1016/0006-2952(91)90595-V Get rights and content

Abstract

There are significant discrepancies in the literature concerning the concentration of monoamine oxidase A (MAO A) from a number of tissue sources. Therefore, we compared the two principal techniques that have been used for quantitation of MAO A protein concentration: (1) titration of the enzyme with the MAO A-selective inhibitor clorgyline, and (2) saturation of the enzyme with [³H]-pargyline followed by immunoprecipitation with an MAO A-specific monoclonal antibody. To determine which of the two techniques was likely to yield more reliable values for MAO A, MAO A protein concentrations in the same preparations were determined by quantitative immunoblotting. [³H]Pargyline binding and quantitative immunoblotting yielded comparable values which were markedly lower than those obtained by titration of MAO A with unlabeled clorgyline. Therefore, clorgyline titration can seriously overestimate the concentration of MAO A protein in mitochondrial preparations. Since many literature values for the molecular activity of MAO A have relied upon enzyme concentrations determined by clorgyline binding, we reevaluated the molecular activities of MAO A and B for five important substrates. The ratio, MAO A molecular activity: MAO B molecular activity decreased in the order: serotonin (35:1)> tryptamine (12:1) >tyramine (3.3:1) > dopamine (2.4:1) > benzylamine (1:23). No comparable ratio was determined for β-phenylethylamine because of its previously described substrate inhibition of MAO B, although it is oxidized faster by MAO B over a wide range of concentrations. Comparison of molecular activities and K_m values for MAO A and B showed that with the exception of benzylamine and β-phenylethylamine, MAO A oxidizes the other tested substrates faster than MAO B over a wide range of concentrations. Therefore, measured ratios of MAO A: MAO B activity are generally greater than the ratios of MAO A:MAO B molecules in the preparations.

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Inhibition of monoamine oxidase attenuates social defeat-induced memory impairment in goldfish, (Carassius auratus): A possible involvement of synaptic proteins and BDNF
2021, Comparative Biochemistry and Physiology Part - C: Toxicology and Pharmacology
Citation Excerpt :
Supporting to our observation, earlier it has been reported that stress elevated the level of MAO-A (Meyer et al., 2006), and pre-treatment of MAOI significantly protect the effect of SD (Song et al., 2013). We have not measured the MAO activity in this study, since variation in the substrate selectivity and preparation of MAO have been very sensitive and known to alter the activity (Riley and Denney, 1991). Earlier studies documented that inhibition of MAO activity may resilience the effect of SD on serotonergic signaling, MAO-A and serotonin transporter (SERT) (Pavlov et al., 2012; Dorfman et al., 2014).
Social defeat (SD) has been implicated in different modulatory effects of physiology and behaviour including learning and memory. We designed an experiment to test the functional role of monoamine oxidase (MAO) in regulation of synaptic transmission, synaptic plasticity and memory in goldfish Carassius auratus. To test this, individuals were divided into three groups: (i) control; (ii) social defeat (SD) group (individuals were subjected to social defeat for 10 min by Pseudotropheus demasoni) and (iii) SD + MAO inhibitor pre-treated group. All experimental groups were subjected to spatial learning and then memory. Our results suggest that SD affects a spatial learning and memory, whereas SD exerts no influence on MAOI pre-treated group. In addition, we noted that the expression of monoamine oxidase-A (MAO-A) was up-regulated and level of serotonin (5-hydroxytryptamine; 5-HT), expression of serotonin transporter (SERT), synaptophysin (SYP), synaptotagmin −1 (SYT-1), N-methyl-D-asparate (NMDA) receptors subunits (NR2A and NR2B), postsynaptic density-95 (PSD-95) and brain-derived neurotrophic factor (BDNF) were reduced by SD, while MAOIs pretreatment protects the effect of SD. Taken together, our results suggest that MAO is an essential component in the serotonergic system that finely tunes the level of 5-HT, which further regulates the molecules involving in synaptic transmission, synaptic plasticity and memory.
Monoamine Oxidase (MAO) in Human Peripheral Tissues
2004, NeuroToxicology
Synthesis and biological evaluation of MAO-A selective 1,4-disubstituted-1,2,3,6-tetrahydropyridinyl substrates
2002, Bioorganic and Medicinal Chemistry
Many mammalian tissues express both the A and B forms of monoamine oxidase (MAO), flavoenzymes that catalyze the oxidative deamination of a variety of endogenous and exogenous amines and the ring α-carbon oxidative bioactivation of neurotoxic 1,4-disubstituted-1,2,3,6-tetrahydropyridinyl derivatives. Substrates selective for MAO-A that display good kinetic and spectroscopic properties would be of value for developing quantitative assays for MAO-A in tissues that express both the A and B forms of the enzyme. This paper describes the synthesis of several 1-substituted-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridinyl derivatives. Kinetic parameters and MAO-A selectivity indicate that 1-allyl- and 1-propyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine should be good candidates to develop a robust spectrophotometric-based assay that is selective for MAO-A.
Localization of I<inf>2</inf>-imidazoline binding sites on monoamine oxidases
1995, Journal of Biological Chemistry
Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, I₁BS and I₂BS. Convergent studies attribute a role in central blood pressure regulation to the I₁BS. In contrast, the function of I₂BS remains unknown.
In the present study, by combining biochemical and molecular biology approaches, we show that 1) microsequencing of I₂BS purified from rabbit kidney mitochondria allowed the recovery of four peptide sequence stretches displaying up to 85.7% similarity with human, rat, and bovine monoamine oxidases (MAO)-A and -B; 2) I₂BS and MAO displayed identical biophysical characteristics as their activities, measured by [³H]idazoxan binding and [¹⁴C]tyramine oxidation, respectively, could not be separated using various chromatographic procedures; and 3) heterologous expression of human placenta MAO-A and human liver MAO-B in yeast, inherently devoid of I₂BS and MAO activities, led to the co-expression of [³H]idazoxan binding sites displaying ligand-recognition properties typical of I₂BS.
These results show definitely that I₂BS is located on both MAO-A and -B. The fact that I₂BS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that I₂BS might be a previously unknown MAO regulatory site.
Elevation of neuronal MAO-B activity in a transgenic mouse model does not increase sensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
1994, Brain Research
To examine whether expressing high levels of monoamine oxidase (MAO-B) activity abberently in neurons results in increased sensitivity of dopaminergic neurons to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 8-week-old transgenic mice expressing high neuronal levels of MAO-B were compared with age-matched nontransgenic littermates following i.p. injections of 30 mg/kg body weight of the protoxin. Levels of striatal dopamine (DA) and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), as well as tyrosine hydroxylase (TH)-immunopositive cell numbers in the substantia nigra (SN) were compared 1 week later between transgenics and controls. No difference was found in any of these parameters, indicating that high neuronal MAO-B levels does not cause increased sensitivity to MPTP, and therefore neither conversion of MPTP to its active form, 1-methyl-4-phenyl pyridium (MPP+) by MAO-B nor MPP+ uptake by the dopaminergic transporter are likely to be the rate-limiting step in the toxicity of this compound.
An examination of the reliability of the radiochemical assay for monoamine oxidases A and B
1993, Analytical Biochemistry
The radiochemical assay for MAO A has been compared with the polarographic and alcohol dehydrogenase-coupled assays, using 5-hydroxytryptamine (5-HT) and tyramine and the homogeneous human liver enzyme expressed in yeast, and rat liver mitochondria. Despite efforts to measure true initial rates and to avoid known sources of error in the radiochemical procedure, significantly higher rates (V_max) and lower K_m values for the substrate were obtained with the polarographic than with the radiochemical method for 5-HT and tyramine, using either highly purified enzyme or mitochondria. The rate of tyramine oxidation measured by the polarographic method and the coupled assay agreed well, however. Consequently, for kinetic and inhibition studies we recommend the polarographic method for MAO A substrates. Comparison of the radiochemical, polarographic, spectrophotometric, and coupled assays for MAO B from beef liver (mitochondria and homogenous enzyme) showed polarography to be the method of choice on all substrates tested, although, if all known sources of error are carefully controlled, the radiochemical assay gives the same V_max and K_m values. We also report that with each substrate studied the apparent K_m is much lower when mitochondria are used than when the highly purified, virtually lipid-free preparations of MAO A are studied.

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^∗: Current address: Leigh A. Riley, Ph.D., Department of Biological Sciences, Smith Hall, Rutgers University, Newark, NJ 07102 USA

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Research paperProblems with the measurement of monoamine oxidase A protein concentration in mitochondrial preparations Revised molecular activities and implications for estimating ratios of MAO A:MAO B molecules from radiochemical assay data

Abstract

Biochem Biophys Res Commun

Biochem Pharmacol

Biochem Pharmacol

Biochem Pharmacol

Psychiatry Res.

J Biol Chem

Biochem Pharmacol

Life Sci

Eur J Pharmacol

Neuroscience

Neuroscience

Amine oxidases and their endogenous substrates

J Neural Transm

An appraisal of the use of monoamine oxidase as an enzyme marker for the outer mitochondrial membrane

J Cell Biol

The catalytic behaviour of monoamine oxidase

J Neural Transm

Multiple binding sites of human brain and liver monoamine oxidase: Substrate specificities, selective inhibitions, and attempts to separate enzyme forms

J Neurochem

Some puzzling pharmacological effects of monoamine oxidase inhibitors

Adv Biochem

Human liver MAO-A and MAO-B separated by immunoaffinity chromatography with MAO-B-specific monoclonal antibody

Science

A monoclonal antibody elicited to human platelet MAO

Mol Pharmacol

Immunological uniqueness of human monoamine oxidases A and B: New evidence from studies with monoclonal antibodies to human monoamine oxidase A

J Neurosci

cDNA cloning of human liver monoamine oxidase A and B: Molecular basis of differences in enzymatic properties

Neurobiology

Research paper
Problems with the measurement of monoamine oxidase A protein concentration in mitochondrial preparations Revised molecular activities and implications for estimating ratios of MAO A:MAO B molecules from radiochemical assay data