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Fe2+ stimulates the binding of [3H]serotonin and [3H]dopamine to rabbit skeletal muscle actin. This binding is inhibited by reducing agents (sodium ascorbate, vitamin E), by superoxide dismutase and by sulfhydryl group-modifying reagents (N-ethyl-maleimide, 2,2'-dinitro-5,5'-dithiobenzoic acid). The effect of Fe2+ is mimicked by oxidants (sodium periodate, potassium nitroso-disulfonate) and by superoxide radicals. Once formed, the binding cannot be decreased by a large excess of monoamine. It is proposed that Fe2+ catalyses the autoxidation of the monoamines by generating oxygen free radicals, and the oxidation products are likely to bind covalently to exposed cysteine residues of actin. Digestion of [3H]dopamine-labelled actin by cyanogen bromide and then by 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 sites on actin. This inactivation, and the covalent nature of the binding precludes the interpretation of monoamine saturation and competition binding data in terms of reversible bimolecular interactions.

Binding of [3H]serotonin and [3H]dopamine to serotonin-binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+. This group recently attributed this effect of Fe2+ to its ability to enhance the oxidation of [3H]serotonin and [3H]dopamine in the presence of dissolved molecular oxygen, and to the ability of the formed oxidation products to bind covalently to cysteine residues of SBP. In this study it is shown that the binding of both ligands is potently inhibited by dopamine as well as by several catecholamine-and serotonin-related neurotoxins: adrenochrome, 5,6-dihydroxytryptamine, 5,7-dihydroxytryptamine, 6-hydroxydopamine and 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline. In contrast, serotonin can only potently inhibit part (36%) of the [3H]dopamine binding, while 1,2,3,4-tetrahydroisoquinoline is only a weak competitor for both ligands. Potent inhibition by the toxins is associated with the presence of electrophilic centres at the aromatic ring, either of the products themselves (adrenochrome) or of their oxidation products (all other competitors). These findings suggest that "SBP" represent an important target for the Fe(2+)-mediated binding of [3H]-serotonin, [3H]dopamine and related neurotoxins.

The soluble serotonin-binding proteins (SBP) present in the adrenal medulla and in chromaffin cells, are very similar to those reported for the bovine brain and retina. Binding of [3H]serotonin and [3H]dopamine to these SBP is increased by Fe2+ but not by Fe3+. At an optimal concentration of Fe2+ (0.1 mM) these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 124 +/- 28 pmol/mg protein, KD = 0.51 +/- 0.13 microM) and [3H]dopamine (Bmax = 685 +/- 118 pmol/mg protein, KD = 0.46 +/- 0.06 microM). Binding of [3H]dopamine is also increased by Cu2+ and Mn2+, but to a lesser extent than by Fe2+. Catecholamines are good competitors for [3H]serotonin binding (Ki = 0.31 microM for dopamine, 0.6 microM for adrenaline and 0.9 microM for noradrenaline). The serotonin binding proteins from adrenal medulla elute in the void volume of a Sephacryl 100 HR gel filtration column, reflecting aggregation, and migrate mainly with an apparent molecular weight of 45 kDa in native polyacrylamide gel electrophoresis experiments. Subcellular localization studies and release experiments suggest that SBP are not present in chromaffin granules, but in the cytosol of purified chromaffin cells. The present data suggest that these proteins must have other functions than storing monoamines in synaptic vesicles.

Binding of [3H]serotonin and of [3H]dopamine to serotonin binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+ but not by Fe3+. It was generally believed that Fe2+ first binds to sulfhydryl groups of SBP and that the monoamines form coordination bonds with the trapped iron. We report two series of findings that are incompatible with this mechanism. First, the binding of both radioligands is an irreversible process since it is not diminished when a large excess (1 mM) of serotonin or dopamine is added to a pre-equilibrated mixture of SBP, 0.1 mM Fe2+ and 0.2 microM radioligand. Once formed, binding is not impaired by chelating agents such as ethyleneglycoltetraacetic acid and desferal. Second, the Fe(2+)-stimulated binding is inhibited by reducing agents (sodium ascorbate, vitamin E, sodium metabisulfite) and by agents which deplete superoxide radicals (superoxide dismutase and hydrogen peroxide). Moreover, the effect of Fe2+ can be mimicked by oxidants (sodium periodate, potassium superoxide) and by the generation of superoxide radicals by the xanthine oxidase-catalysed oxidation of xanthine. To integrate these findings, we formulate the hypothesis that Fe2+ reacts with dissolved molecular oxygen to produce superoxide radicals, that these radicals oxidise [3H]serotonin and [3H]dopamine, and that the formed oxidation products bind covalently to cysteine residues of SBP. This alternative mechanism is also based on the ability of reagents which contain or modify sulfhydryl groups to decrease the binding and on the inability of hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol and thiourea) to do so. Fe2+ is also able to irreversibly inactivate part of the binding sites on SBP (81% of the specific binding of [3H]serotonin, and 61% for [3H]dopamine). This Fe(2+)-mediated inactivation, as well as the covalent nature of the binding, preclude the interpretation of saturation and competition binding data in terms of reversible bimolecular interactions. Yet, such experiments indicate that, at the same concentration, [3H]dopamine binds to 2 to 3 times more sites than [3H]serotonin. Unlabelled dopamine acts also as a potent competitor at all the [3H]serotonin binding sites, whereas unlabelled serotonin only acts as a potent competitor at part (30%) of the [3H]dopamine binding sites. SBP were initially proposed to be involved in the storage, protection and/or transport of serotonin, and recently also of catecholamines. However, these potential functions of SBP can hardly be reconciled with the molecular mechanism of the binding. Moreover, it is conceivable that this binding actually represents an in vitro model for neurodegeneration.

Serotonin binding proteins (SBP) are present in the soluble fraction of bovine retina homogenates. These proteins can be precipitated with 30% ammonium sulphate and their binding and physicochemical characteristics are very similar to those of SBP in bovine and rat brain. Binding of [3H]serotonin to bovine retina SBP requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 242 +/- 10 pmol/mg protein, KD = 0.22 +/- 0.44 microM). Competition binding studies reveal that serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are potent competitors (K1 from 0.12 to 0.3 microM). In both cases, the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine SBP interactions can also be demonstrated directly by binding experiments with [3H]dopamine. Binding of this catecholamine is greatly enhanced by Fe2+, to a lesser extent by Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component is saturable (Bmax = 505 +/- 30 pmol/mg protein. KD = 0.34 +/- 0.04 microM). The SBP from bovine retina show the same physicochemical properties as SBP from bovine and rat brain: they elute immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and they migrate with apparent molecular weights of respectively 43 kDa and 57 kDa on native polyacrylamide gel electrophoresis. The serotonin-storing role of SBP in serotonergic neurones has already been well documented.(ABSTRACT TRUNCATED AT 250 WORDS)

The soluble serotonin-binding proteins (SBP) present in bovine frontal cortex are very similar to those reported in rat brain. Binding of [3H]serotonin to SBP, present in ammonium sulphate-precipitated proteins from bovine cortex, requires Fe2+ but not Fe3+. In the presence of an optimal concentration of Fe2+ (0.1 mM), bovine SBP behave as a single class of non-cooperative sites for [3H]serotonin binding (Bmax = 120 +/- 12 pmol/mg protein, KD = 0.12 +/- 0.04 microM, n = 3). Binding of [3H]serotonin is decreased by nucleotides and by reagents which modify sulfhydryl groups and reduce disulfide bonds and by metal ion chelators. Serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are effective competitors (Ki from 0.1 to 0.3 microM). In both cases, the aliphatic amino group does not contribute to the binding, but the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments. Binding of [3H]dopamine is greatly enhanced by Fe2+, Cu2+ and Mn2+, but not by Fe3+. The Fe(2+)-dependent binding component of [3H]dopamine is saturable (Bmax = 279 +/- 64 pmol/mg protein, KD = 0.19 +/- 0.02 microM, n = 3), and possesses the same physicochemical properties as SBP: it elutes immediately after the void volume on a Sephacryl S100 HR (1.6 x 140 cm) gel filtration column (reflecting aggregation) and it migrates with an apparent molecular weight of 57-58 kDa on native polyacrylamide gel electrophoresis. Whereas the serotonin-storing role of SBP in serotonergic neurons has already been well documented, the present data advocate that these proteins may also possess catecholamine-storing properties.