FE2-MEDIATED BINDING OF SEROTONIN AND DOPAMINE TO SKELETAL-MUSCLE ACTIN - RESEMBLANCE TO SEROTONIN BINDING-PROTEINS()

Fe2+ stimulates the binding of [H-3]serotonin and [H-3]dopamine to rab bit skeletal muscle actin. This binding is inhibited by reducing agent s (sodium ascorbate, vitamin E), by superoxide dismutase and by sulfhy dryl group-modifying reagents (N-ethylmaleimide, 2,2'-dinitro-5,5'-dit hiobenzoic acid). The effect of Fe2+ is mimicked by oxidants (sodium p eriodate, potassium nitroso-disulfonate) and by superoxide radicals. O nce formed, the binding cannot be decreased by a large excess of monoa mine. It is proposed that Fe2+ catalyses the autoxidation of the monoa mines by generating oxygen free radicals, and the oxidation products a re likely to bind covalently to exposed cysteine residues of actin. Di gestion of [H-3]dopamine-labelled actin by cyanogen bromide and then b y V8 protease(EC3.4.21.19) yields two labelled peptides whose apparent molecular weights (4.1 and 1.2 kDa) are compatible with the labelling of cysteine-10 and -374. Fe2+ also inactivates some of the binding si tes of actin. This inactivation, and the covalent nature of the bindin g precludes the interpretation of monoamine saturation and competition binding data in terms of reversible bimolecular interactions.