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Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis.

ABSTRACT: TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to ?-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by ?-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.

SUBMITTER: Wang J

PROVIDER: S-EPMC3068223 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis.

Wang Jin J Edmondson Dale E DE

Biochemistry 20110307 13

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amid ...[more]

PMID: 21341713

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Project description:This paper reports on dihydroxycoumarins as fluorescent probes suitable for the detection and determination of the nitroxide radical, namely 4-amino-TEMPO. Since 4-amino-TEMPO is used as a spin label for the detection of various radicals and damage caused by these species, its determination under physiological conditions might help us to understand the mechanism of the oxidative stress. Among different coumarins studied, only dihydroxy-substituted derivatives show high sensitivity, specificity, and selectivity for the nitroxide radical. In this assay, dihydroxy-substituted coumarins under the action of 4-amino-TEMPO show a very fast and significant increase in fluorescence intensity and lifetime. Among them 6,7-dihydroxycoumarin (esculetin) exhibits the strongest fluorescence enhancement (up to 40 times), with an estimated limit of detection equal to 16.7 nM-a significantly lower value when compared with UV-Vis or electron paramagnetic resonance (EPR) spectroscopy. The method is characterized by an easy procedure of sample preparation and very short time of analysis. The mechanism of the interaction between 6,7-dihydroxycoumarin and 4-amino-TEMPO has been examined with the use of a series of complementary techniques, such as steady-state and time-resolved fluorescence spectroscopy, UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy, potentiometric titration, and high-performance liquid chromatography. It has been proven that the only route of the reaction in the system studied is a proton transfer from the molecule of esculetin to the amino group of the nitroxide. Biological studies performed on prostate cancer cells, breast cancer cells, and normal skin fibroblasts revealed significant anticancer properties of 6,7-dihydroxycoumarin, which caused a considerable decrease in the viability and number of cancer cells, and affected their morphology, contrary to normal fibroblasts. Furthermore, the experiment performed on prostate cancer cells showed that fluorescence emission of esculetin is closely related to intracellular pH-the higher pH, the higher observed fluorescence intensity (in accordance with a chemical experiment). On the other hand, the studies performed in different pH levels revealed that when pH of the solution increases, the observed fluorescence intensity enhancement under the action of 4-amino-TEMPO decreases (better sensing properties of esculetin towards the nitroxide in lower pH).

Project description:Background: Monoamine oxidases (MAOs) were discovered nearly a century ago. This article aims to analyze the research literature landscape associated with MAOs as privileged class of neuronal enzymes (neuroenzymes) with key functions in the processes of neurodegeneration, serving as important biological targets in neuroscience. With the accumulating publications on this topic, we aimed to evaluate the publication and citation performance of the contributors, reveal the popular research themes, and identify its historical roots. Methods: The electronic database of Web of Science (WoS) Core Collection was searched to identify publications related to MAOs, which were analyzed according to their publication year, authorship, institutions, countries/regions, journal title, WoS category, total citation count, and publication type. VOSviewer was utilized to visualize the citation patterns of the words appearing in the titles and abstracts, and author keywords. CRExplorer was utilized to identify seminal references cited by the MAO publications. Results: The literature analysis was based on 19,854 publications. Most of them were original articles (n = 15,148, 76.3%) and reviews (n = 2,039, 10.3%). The top five WoS categories of the analyzed MAO publications were Pharmacology/Pharmacy (n = 4,664, 23.5%), Neurosciences (n = 4,416, 22.2%), Psychiatry (n = 2,906, 14.6%), Biochemistry/Molecular Biology (n = 2,691, 13.6%), and Clinical Neurology (n = 1,754, 8.8%). The top 10 institutions are scattered in the United States, UK, France, Sweden, Canada, Israel, and Russia, while the top 10 countries/regions with the most intensive research on the field of MAOs are the United States, followed by European and Asian countries. More highly cited publications generally involved neurotransmitters, such as dopamine (DA), serotonin, and norepinephrine (NE), as well as the MAO-A inhibitors moclobemide and clorgyline, and the irreversible MAO-B inhibitors selegiline and rasagiline. Conclusion: Through decades of research, the literature has accumulated many publications investigating the therapeutic effects of MAO inhibitors (MAOIs) on various neurological conditions, such as Alzheimer's disease (AD), Parkinson's disease (PD), and depression. We envision that MAO literature will continue to grow steadily, with more new therapeutic candidates being tested for better management of neurological conditions, in particular, with the development of multi-target acting drugs against neurodegenerative diseases.

Dataset Information

Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis.

Publications

Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis.

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