title: Calcium-dependent calmodulin binding to cholinergic synaptic vesicles.
abstract: Calmodulin is a major nerve terminal protein and a potential mediator of calcium-dependent nerve terminal functions. Calcium-dependent calmodulin binding has been reported in secretory membrane preparations including chromaffin granules and crude rat brain vesicles. Here we demonstrate a calcium-dependent calmodulin-binding site on cholinergic synaptic vesicles from electric organ. It is saturable with high affinity (KD = 10 nM; Bmax = 80 pmol/mg). The binding is inhibited by trifluoperazine (I50 = 8 microM) and is at least 1000-fold specific for calmodulin over troponin C. Association and dissociation rates (k = 3.1 X 10(6) M-1S-1; k-1 = 1.3 X 10(-2) S-1) are consistent with the dissociation constant measured at equilibrium. Intact synaptic vesicles bind to calmodulin immobilized on polyacrylamide matrix, suggesting that the binding site is cytoplasmically oriented in the vesicle population. Intact synaptic vesicles bind calmodulin up to 80-fold more effectively than do side fractions from the vesicle purification. The quantitative difference is largely due to latency of binding sites since it disappears when the binding is assayed in detergent. Binding of calmodulin to proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that a subset of nerve terminal and electric organ calmodulin-binding proteins are found in synaptic vesicles.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism*; D014101:Torpedo; D014268:Trifluoperazine / Q000494:pharmacology; D014336:Troponin / Q000378:metabolism; D019209:Troponin C
chemical_list: D002147:Calmodulin; D011950:Receptors, Cholinergic; D014336:Troponin; D019209:Troponin C; D014268:Trifluoperazine; D000255:Adenosine Triphosphate; D002118:Calcium