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 membranebound enzymes are located on differing faces of the mitochondrial outer membrane (Upadhyay, A. and Edmondson, D.E., Biochemistry 48, 3928, 2009). 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 meta-and para-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 opposite inhibition pattern 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 β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast on trypsin treatment and MAO B is readily inactivated by β-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 neuro-protectants.The known age-related increases in expression of Monoamine Oxidase B (MAO B) 1 in neuronal tissue (1) and Monoamine Oxidase A (MAO A) in heart (2) have been implicated in neurological (3) and cardiovascular disorders (4). Design of highly specific reversible inhibitors for each enzyme that could serve as neuro-protectants and cardio-protectants has been and is currently receiving increased attention. It is known that MAO A and MAO B levels vary among different tissues (5). In all cases, both enzymes are dimeric (6) and found tightly bound to the outer membrane of the mitochondrion via C-terminal trans-membrane helices as well as undetermined membrane interactions with the core polypeptide chain (7,8). † This work was supported by National Institute of Health Grant GM-29433 (DEE).* To whom correspondence should be addressed. Dr. Dale E. Edmondson: Department of Biochemistry, Rollins Research Bldg. Emory University, 1510 Clifton Rd. Atlanta, GA 30322; deedmon@emory.edu; Phone: 404-727-5972; Fax: 404-727-2738. Supporting Information Available: The structures of the TEMPO-substituted pargyline analogues used in this study are shown. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations: MAO: Monoamine Oxidase; MOM: mitochondrial outer membrane; TEMPO: 2,2,6,6-tetramethylpiperidinyl-1-oxyl; ParSL: spin-lab...

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