Proteases as probes of mitochondrial monoamine oxidase topography in situ.

Selective inactivation of the multiple forms of mitochondrial monoamine oxidase (MAO) by proteases in intact and hypotonically disrupted rat liver mitochondria has been used to examine the question of differential membrane orientations of the A and B enzymes. Proteases used as probes included trypsin, beta-chymotrypsin, and the extracellular protease of Staphylococcus aureus, chosen for their different amino acid specificities. With all three proteases, no changes in the relative rates of MAO-A and MAO-B inactivation were observed after disruption of the mitochondria. Trypsin and beta-chymotrypsin gave much faster rates of MAO-A inactivation in both intact and disrupted mitochondria. The selective effect of trypsin on MAO-A was also confirmed in human placental mitochondria, which possess only A-type activity. The effectiveness of hypotonicity in disrupting the outer membrane of the mitochondria was shown by rapid protease inactivation of an intermembrane space marker enzyme, adenylate kinase (EC 2.7.4.3). Contrary to some recent reports in the literature, these findings strongly suggest that the MAO-A and MAO-B multiple-form catalytic activities do not reside on opposite faces of the membrane.