Detection by chemical cross-linking of bovine brain synapsin I self-association.
Synapsin I is believed to play an important role in the regulation of neurotransmitter release, since it is able to bind to synaptic vesicles, to the cytoskeleton and to membrane proteins; in addition, it bundles F-actin and microtubules. These properties, which are controlled by phosphorylation, could be explained if synapsin has different and multiple binding sites or if synapsin I is able to form polymers by self-association. In this study we present experimental evidence that synapsin I at low concentration forms self-associated dimers, as revealed after mild treatments with cross-linking agents. We have especially studied here the effects of copper/o-phenanthroline, a zero-length cross-linking agent which forms covalent links by oxidative formation of S-S bridges between adjacent cysteines. The time course and concentration-dependence of synapsin-dimer formation are studied; interestingly, these experiments could suggest a different behaviour of the two polypeptides. Limited proteolysis of phosphorylated synapsin I by V8 protease, alpha-chymotrypsin or collagenase, performed on the isolated dimer and monomer, allows us to localize tentatively in the central hydrophobic core of the molecule the cysteine residues the oxidation of which by copper/o-phenanthroline gives rise to synapsin dimers.
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LBTM-CNRS, Université Claude Bernard Lyon I, Villeurbanne, France.
Aubert-Foucher
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D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003300:Copper / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010618:Phenanthrolines / Q000494:pharmacology*; D010766:Phosphorylation; D016704:Synapsins
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010618:Phenanthrolines; D016704:Synapsins; D003300:Copper; C025205:1,10-phenanthroline