Evidence for the binding of calmodulin to endogenous B-50 (GAP-43) in native synaptosomal plasma membranes.
The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.
De Graan
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PN
Division of Molecular Neurobiology, Rudolf Magnus Institute, Medical Faculty, University of Utrecht, The Netherlands.
Oestreicher
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AB
De Wit
M
M
Kroef
M
M
Schrama
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LH
Gispen
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D000818:Animals; D015153:Blotting, Western; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D013388:Succinimides; D013574:Synaptosomes / Q000378:metabolism*
D002147:Calmodulin; D003432:Cross-Linking Reagents; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D013388:Succinimides; C019358:disuccinimidyl suberate