title: [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 for [3H]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 [3H]Ro 16-6491. The density of the sites labelled with high affinity by [3H]Ro 19-6327 was similar to that observed in previous studies with [3H]Ro 16-6491 as ligand. Binding was reversible at 20 degrees C and showed a relatively slow dissociation (t1/2 = 220 min). The dissociation rate was markedly decreased (t1/2 = greater than 24h) at 0 degrees C. MAO-B, but not MAO-A inhibitors, effectively prevented the binding of [3H]Ro 19-6327. Like [3H]Ro 16-6491, [3H]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 [3H]Ro 19-6327. Incubation of the radioligand-enzyme complex from platelet and brain membranes with NaBH3CN 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 [3H]Ro 19-6327. The irreversible MAO-B inhibitor, [3H] pargyline, labelled a protein with a molecular weight identical to the protein labelled by [3H]Ro 19-6327.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Pharmacology, University of Milan, Italy.
authors.forename: A M
authors.initials: AM
authors.lastname: Cesura
authors.forename: M D
authors.initials: MD
authors.lastname: Galva
authors.forename: R
authors.initials: R
authors.lastname: Imhof
authors.forename: E
authors.initials: E
authors.lastname: Kyburz
authors.forename: G B
authors.initials: GB
authors.lastname: Picotti
authors.forename: M
authors.initials: M
authors.lastname: Da Prada
mesh_terms: D000345:Affinity Labels; D001549:Benzamides / Q000378:metabolism; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism; D001921:Brain / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D066298:In Vitro Techniques; D008995:Monoamine Oxidase / Q000378:metabolism; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010848:Picolinic Acids / Q000494:pharmacology*
chemical_list: D000345:Affinity Labels; D001549:Benzamides; D008996:Monoamine Oxidase Inhibitors; D009419:Nerve Tissue Proteins; D010848:Picolinic Acids; C059303:lazabemide; C051040:Ro 16-6491; D008995:Monoamine Oxidase