[3H]Ro 19-6327: A reversible ligand and affinity labelling probe for monoamine oxidase-B

Abstract This study demonstrated the existence of specific binding sites [ 3 H]Ro 19-6327 in human platelet membranes. This compound is a novel, time-dependent inhibitor of monoamine oxidase type B (MAO-B) and is structurally closely related to [ 3 H]Ro 16-6491. The density of the sites labelled with high affinity by [ 3 H]Ro 19-6327 was similar to that observed in previous studies with [ 3 H]Ro 16-6491 as ligand. Binding was reversible at 20°C and showed a relatively slow dissociation ( t 1 2 =220 min ) . THe dissociation rate was markedly decreased ( t 1 2 = > 24 h ) at 0 °C. MAO-B, but not MAO-A inhibitors, effectively prevented the binding of [ 3 H]Ro 19-6327. Like [ 3 H]Ro 16-6491, [ 3 H]Ro 19-6327 is recognized as a substrate by MAO-B, being eventually deaminated by the enzyme. Since the deaminated aldehyde derivative of Ro 19-6327 did not inhibit MAO-B, a still unidentified reversible adduct, formed at the MAO-B active site, might explain the high potency and selectivity of [ 3 H]Ro 19-6327. Incubation of the radioligand-enzyme complex from platelet and brain membranes with NaBH 3 CN and acetic acid (to pH 4.5) caused the irreversible incorporation of the radioactivity into a single polypeptide as shown by SDS-PAGE analysis. This polypeptide had a molecular weight identical to that of the MAO-B subunit, i.e. 58 000. The presence of unlabelled MAO-B inhibitors in the incubation mixture prevented the covalent incorporation of [ 3 H]Ro 19-6327. The irreversible MAO-B inhibitor, [ 3 H] pargyline, labelled a protein with a molecular weight identical to the protein labelled by [ 3 H]Ro 19-6327. These data indicate that [ 3 H]Ro 19-6327 in the presence of NaBH 3 CN is irreversibly incorporated into one of the two polypeptide subunits of MAO-B. Therefore, [ 3 H]Ro 19-6327 can also be used as an irreversible affinity-labelling probe for MAO-B. Since both Ro 19-6327 and Ro 16-6491 inhibit MAO-B irreversibly in the presence of NaBH 3 CN these compounds might be very useful for the localization and identification of the active site of the enzyme.