The inhibition of rat liver mitochondrial monoamine oxidase-A (MAO-A) by brofaromine was time-dependent at low enzyme and inhibitor concentrations. The apparent sensitivity to inhibition decreased when the concentration of the mitochondrial preparation was increased. After preincubation of the enzyme with brofaromine repeated washing of the preparation, by sedimentation and resuspension, resulted in a gradual recovery of activity. This occurred more slowly than was the case when the reversible inhibitor amphetamine was used. After incubation with radioactively-labeled brofaromine the loss of radioactivity also occurred slowly. After incubation with radioactively-labeled pargyline polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphage (SDS-PAGE) showed the radioactivity to be associated with a peptide of approximate Mr 50,000, corresponding to the subunit of MAO. Pretreatment with unlabeled pargyline depressed this labeling by pargyline, indicating the latter compound to bind to the active-site of the enzyme. Labeling experiments with radioactive brofaromine indicated that there was a high degree of non-specific binding but that no significant radioactivity remained associated with the enzyme on SDS-PAGE. Chromatographic techniques and determination of H2O2 liberation indicated that, in liver there was no appreciable metabolism of brofaromine under the conditions used in the inhibition experiments. These data indicate brofaromine to be a tight-binding, but reversible inhibitor of MAO.
