PubMed · 14082231
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D Y HSIA, K NISHIMURA, Y BRENCHLEY. 1963-11-09. MECHANISMS FOR THE DECREASE OF BRAIN SEROTONIN.. https://doi.org/10.1038/200578a0
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1. Binding of the novel radioligand (3)H-2-(2-dimethylaminomethyl-phenylsulphanyl)-5-methyl-phenylamine ((3)H-MADAM) to the serotonin transporter (SERT) was used to characterise a range of selective serotonin re-uptake inhibitors (SSRIs) in vitro and in vivo. 2. (3)H-MADAM bound with high affinity in a saturable manner to both human SERT expressed in CHO cells (K(d)=0.20 nm (pK(d)=9.74+/-0.12), B(max)=35+/-4 fmol mg(-1) protein) and mouse cerebral cortex membranes (K(d)=0.21 nm (pK(d)=9.66+/-0.10), B(max)=50+/-24 fmol mg(-1) protein). 3. Binding of (3)H-MADAM was highly selective for SERT in vitro as demonstrated by the in vitro profile of MADAM tested at 75 different receptors, ion channels and transporters. This was further substantiated by the pharmacological profile of the binding. Hence, the binding of (3)H-MADAM was potently inhibited by SSRIs but not by selective inhibitors of noradrenaline transport and dopamine transport. Likewise, a 5-HT(2A/2C) receptor antagonist did not inhibit (3)H-MADAM binding. 4. (3)H-MADAM binding in vivo was inhibited only by compounds which also inhibited the binding of (3)H-MADAM in vitro (the SSRIs, mixed SERT/noradrenaline transport inhibitors and clomipramine), confirming the selectivity of (3)H-MADAM for SERT also in vivo. Moreover, compounds effective in inhibiting (3)H-MADAM binding were the only ones found to be active in the mouse 5-HTP potentiation test confirming the model as a behavioural correlate to in vivo 5-HT uptake. 5. Finally, it was found that a SERT occupancy of 85-95% was necessary to produce 50% of the maximum behavioural response (ED(50)).
l-5-hydroxytryptophan (5-HTP) with two types of multiple (19)F-atom tags bonded at various positions onto the indole ring (positions 4, 6, or 7) was exposed to aromatic l-amino acid decarboxylase (AADC) in lysates of Escherichia coli JM109 which had been transformed with the plasmid pKKAADCII. Resulting samples were analyzed with HPLC. In the first study, which investigated a straight-chain seven-atom tag, a novel peak, putatively perfluoro-tagged serotonin, was detected. A second study demonstrated that 5-HTP was converted to 5-HT in transformed E.coli lysate but not in untransformed lysate. A third study, investigating a tag with nine fluorine atoms all in the same nuclear environment, identified the isomer serving as the best substrate for AADC. This novel molecule had the tag bonded at the 6 position on the indole ring. Isomers that fit into the active site of AADC are likely to follow the biosynthetic path for serotonin in vivo and are potentially useful in (19)F magnetic resonance spectroscopy studies. The enzymatic assay described here provides an efficient and cost-effective tool for screening new compounds.
Tropomyosin is a filamentous coiled-coil protein directly involved in the regulation of the actomyosin interaction responsible for muscle contraction: it transmits the local calcium-induced conformational change in troponin to the helical array of myosin-binding sites on the surface of the actin filament. McLachlan and Stewart (McLachlan, A. D., and Stewart, M. (1976) J. Mol. Biol. 103, 271-298) proposed that the tropomyosin coiled-coil structure can be divided into 14 alternating 19- to 20-residue "alpha- and beta-bands," which could act as alternate 7-fold sets of sites for specific binding to actin in the different conformational states of the regulated thin filament. Here we present the first direct experimental evidence in support of the alpha- and beta-band hypothesis: we analyze the acrylamide quenching of the fluorescence of mutant tropomyosins containing 5-hydroxytryptophan residues at different positions along the coiled-coil structure under a variety of conditions (alone, complexed with actin, and complexed with actin and troponin with or without Ca(2+)). We show that fluorescent probes placed in the alpha-bands become less solvent-exposed in the absence of calcium, whereas those in the beta-bands become less solvent-exposed in the presence of calcium. A model in which the tropomyosin coiled-coil rolls across the actin surface in response to Ca(2+)-binding to troponin most easily explains these observations.