Preliminary characterization of synaptic vesicle/calmodulin interaction.
The association of calmodulin with brain synaptic vesicle proteins was analyzed. Scatchard analysis of [125I]calmodulin binding to brain synaptic vesicles revealed one high-affinity, low-binding-capacity, Kd = 1.0 (+/- 0.15) nM, Bmax = 4.1 (+/- 0.6) pmol/mg, and one low-affinity high-binding-capacity site, Kd = 177. (+/- 12.0) nM and Bmax = 202 (+/- 15.0) pmol/mg. Triton X-100 solubilization of synaptic vesicle proteins and subsequent elution on a Sepharose-4B-CNBr-calmodulin affinity column demonstrated that two protein doublets of approximate MrS 55 K and 30 K were the major synaptic vesicle calmodulin binding proteins. In addition there were two minor calmodulin binding singlet polypeptides with MrS 62 K and 40 K. Calmodulin stimulated endogenous synaptic vesicle protein kinase, Ca2+, Mg2+-ATPase and Ca2+ uptake activities. Phosphorylation assays coupled with immunological studies using affinity-purified antibodies suggested that the synaptic vesicle Ca2+/calmodulin-dependent protein kinase migrated in the 30 K Mr region.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism* / Q000502:physiology; D002417:Cattle; D002846:Chromatography, Affinity; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D013572:Synaptic Vesicles / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure
D000906:Antibodies; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D009419:Nerve Tissue Proteins; D002118:Calcium