Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Assembly of the three neuronal membrane proteins synaptobrevin, syntaxin, and SNAP-25 is thought to be one of the key steps in mediating exocytosis of synaptic vesicles. In vivo and in vitro, these proteins form a tight complex. Assembly is associated with a large increase in alpha-helical content, suggesting that major structural and conformational changes are associated with the assembly reaction. Limited proteolysis by trypsin, chymotrypsin, and proteinase K of the ternary complex formed from recombinant proteins lacking their membrane anchors revealed a SDS-resistant minimal core. The components of this core complex were purified and characterized by N-terminal sequencing and mass spectrometry. They include a slightly shortened synaptobrevin fragment, C- and N-terminal fragments of SNAP-25, and a C-terminal fragment of syntaxin that is slightly larger than the previously characterized H3 domain. Recombinant proteins corresponding to these fragments are sufficient for assembly and disassembly. In addition, each of the two SNAP-25 fragments can individually form complexes with syntaxin and synaptobrevin, suggesting that they both contribute to the assembly of the SNARE complex. Upon complex assembly, a large increase in alpha-helical content is observed along with a significantly increased melting temperature (Tm). Like the full-length complex, the minimal complex tends to form an oligomeric species; global analysis of equilibrium ultracentrifugation data suggests a monomer-trimer equilibrium exists. These conserved biophysical properties may thus be of fundamental importance in the mechanism of membrane fusion.
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PMID:Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly. 967 3

We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-beta, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-alpha. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-beta exon in the nervous system. Synaptotagmin I C2B-beta showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-alpha, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-beta with a higher affinity than synaptotagmin I C2B-alpha. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.
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PMID:Identification and characterization of a novel C2B splice variant of synaptotagmin I. 1505 79