Effects of lanthanum and trifluoperazine on [125I]calmodulin binding to rat striatal particulates.
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Abstract:
A calmodulin-binding assay was established in rat striatal particulates which were depleted of endogenous calcium and calmodulin by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment. The binding of 125I-labeled calmodulin to this preparation was saturable and time-dependent. The dependence of calmodulin binding upon temperature and concentration was also demonstrated. Calcium was a prerequisite for calmodulin binding and it facilitated the binding in a dose-dependent manner. Lanthanum, a known calcium antagonists in other tissue systems, mimicked the effect of calcium on calmodulin binding. When both ions were present at low concentrations, their effects on calmodulin binding was additive. Lanthanum, but not calcium, inhibited calmodulin release from a non-EGTA-treated preparation. This difference in action between calcium and lanthanum suggests that they mediate calmodulin binding in an independent manner. Scatchard analysis of calcium-calmodulin binding to rat striatal particulates revealed that there are two populations of binding sites: a higher affinity (apparent KD = 1.3 X 10(-7) M) and a lower affinity (apparent KD = 2.9 X 10(-7) M) binding site. Trifluoperazine, a phenothiazine antipsychotic drug, at 10(-4) M antagonized calmodulin binding only at the higher affinity binding sites. These sites may play an important role in mediating the action of trifluoperazine in the caudate nucleus.Keywords:
Trifluoperazine
EGTA
Lanthanum
We have previously reported that the actin gelation inhibitor, derived from platelets is a protein of MW 90, 000.By affinity chromatogaphy in the presence of 0.1mM CaCl2, a protein of MW 20, 000 was obtained in addition to the gelation inhibitor (MW 90, 000) and actin (MW 45, 000). As the protein of MW 20, 000 was gel-electrophoresised in accordance with brain calmodulin in the presence of EGTA or CaCl2, it was thought to be calmodulin. But in the presence of CaCl2, the brain calmodulin could not activate the gelation inhibitor activity. On the other hand when, in the presence of EGTA, the sample containg the gelation inhibitor was mixed with [3H] calmodulin and then chromatographied by Sephacryl S-200gel filtration, the radioactivity of the isotope of calmodulin was detected in accordance with the protein peak of the sample. These results suggest the possibility that the actin gelation inhibitor binds to calmodulin in the presence of EGTA, not in the presence of CaCl2.
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The activity of the Ca pump of inside-out vesicles of human erythrocyte membranes was studied using /sup 45/Ca and membrane filters. It was found that trifluoperazine completely inhibits the increase in the maximum activity of the Ca pump caused by the addition of calmodulin and has no effect on the calmodulin-stimulated increase in the affinity of the Ca pump for Ca/sup 2 +/. A comparison of characteristic curves of the calmodulin-stimulated components of the activity of the Ca pump, inhibited and not inhibited by trifluoperazine, and the fluorescence intensity of N-phenyl-1-naphthylamine in the presence of calmodulin showed that the mechanisms of action of calmodulin on the maximum activity of the Ca pump and its affinity for Ca/sup 2 +/ differ significantly. In the first case the activation was due to the Ca-calmodulin complex and in the second to the calcium-free form of calmodulin. This conclusion is supported by data on the dependence of the activity of the Ca pump on the calmodulin concentration at low and saturating Ca/sup 2 +/ concentrations as well as by the results obtained in the case of moderate treatment of the membranes with trypsin.
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The modulatory action of Ca2+-calmodulin on multiple targets is inhibited by trifluoperazine, which competes with target proteins for calmodulin binding. The structure of calmodulin crystallized with two trifluoperazine molecules is determined by X-ray crystallography at 2.74 Å resolution. The X-ray data together with the characteristic and distinct signals obtained by circular dichroism in solution allowed us to identify the binding domains as well as the order of the binding of two trifluoperazine molecules to calmodulin. Accordingly, the binding of trifluperazine to the C-terminal hydrophobic pocket is followed by the interaction of the second drug molecule with an interdomain site. Recently, we demonstrated that the two bisindole derivatives, vinblastine and KAR-2 [3' '-(β-chloroethyl)-2' ',4' '-dioxo-3,5' '-spirooxazolidino-4-deacetoxyvinblastine], interact with calmodulin with comparable affinity; however, they display different functional effects [Orosz et al. (1997) British J. Pharmacol. 121, 955−962]. The structural basis responsible for these effects were investigated by circular dichroism and fluorescence spectroscopy. The data provide evidence that calmodulin can simultaneously accommodate trifluoperazine and KAR-2 as well as vinblastine and KAR-2, but not trifluoperazine and vinblastine. The combination of the binding and structural data suggests that distinct binding sites exist on calmodulin for vinblastine and KAR-2 which correspond, at least partly, to that of trifluoperazine at the C-terminal hydrophobic pocket and at an interdomain site, respectively. This structural arrangement can explain why these drugs display different anticalmodulin activities. Calmodulin complexed with melittin is also able to bind two trifluoperazine molecules, the binding of which appears to be cooperative. Results obtained with intact and proteolytically cleaved calmodulin reveal that the central linker region of the protein is indispensable for simultanous interactions with two molecules of either identical or different ligands.
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A calmodulin-binding assay was established in rat striatal particulates which were depleted of endogenous calcium and calmodulin by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment. The binding of 125I-labeled calmodulin to this preparation was saturable and time-dependent. The dependence of calmodulin binding upon temperature and concentration was also demonstrated. Calcium was a prerequisite for calmodulin binding and it facilitated the binding in a dose-dependent manner. Lanthanum, a known calcium antagonists in other tissue systems, mimicked the effect of calcium on calmodulin binding. When both ions were present at low concentrations, their effects on calmodulin binding was additive. Lanthanum, but not calcium, inhibited calmodulin release from a non-EGTA-treated preparation. This difference in action between calcium and lanthanum suggests that they mediate calmodulin binding in an independent manner. Scatchard analysis of calcium-calmodulin binding to rat striatal particulates revealed that there are two populations of binding sites: a higher affinity (apparent KD = 1.3 X 10(-7) M) and a lower affinity (apparent KD = 2.9 X 10(-7) M) binding site. Trifluoperazine, a phenothiazine antipsychotic drug, at 10(-4) M antagonized calmodulin binding only at the higher affinity binding sites. These sites may play an important role in mediating the action of trifluoperazine in the caudate nucleus.
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EGTA
Lanthanum
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