title: 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 β-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 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 neuroprotectants.
authors.affiliation: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, United States.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.forename: Dale E
authors.initials: DE
authors.lastname: Edmondson
mesh_terms: D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008995:Monoamine Oxidase / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010293:Pargyline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000378:metabolism; D010880:Piperidines / Q000737:chemistry*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013045:Species Specificity; D013113:Spin Labels; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: C528764:2,2,6,6-tetramethyl-1-piperidine; D008996:Monoamine Oxidase Inhibitors; D010880:Piperidines; D011994:Recombinant Proteins; D013113:Spin Labels; D010293:Pargyline; D008995:Monoamine Oxidase; D002918:Chymotrypsin; D014357:Trypsin