Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis Jin Wang Dale E. Edmondson 10.1021/bi101722b.s001 https://acs.figshare.com/articles/journal_contribution/Topological_Probes_of_Monoamine_Oxidases_A_and_B_in_Rat_Liver_Mitochondria_Inhibition_by_TEMPO_Substituted_Pargyline_Analogues_and_Inactivation_by_Proteolysis/2669518 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) <i>Biochemistry 48</i>, 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 <i>m-</i> and <i>p</i>-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 <i>Pichia pastoris</i> 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. 2011-04-05 00:00:00 rat liver rat MAO B yeast mitochondria MAO B activity Rat Liver Mitochondria Protease inactivation studies show specificity data show MAO rat liver mitochondria Intact rat liver mitochondria exhibit MAO B rat enzymes