Document Detail

A new photoprobe for studying biological activities of secreted phospholipases A2.
MedLine Citation:
PMID:  17303214     Owner:  NLM     Status:  MEDLINE    
Ammodytoxin (Atx) is a snake venom phospholipase A2 (sPLA2s) with presynaptic toxicity, anticoagulant activity and the ability to influence cell cycle progression. These multiple physiological activities make this molecule a promising tool for studying processes influenced by the highly homologous mammalian sPLA2s-for example cell proliferation and apoptosis. Secreted PLA2s can act on cells as enzymes or as ligands for cellular receptors. To further characterize the sPLA2-binding molecules in cells we have developed a new method based on AtxC and a biotin-containing cross-linking reagent sulfo-SBED which possesses both an amine-reactive and a photo-reactive site, together with a biotin moiety that enables specific detection and affinity-based concentration. The biological activity of the AtxC derivatized by sulfo-SBED was demonstrated by biotin-tagging of calmodulin and R25, both known AtxC targets, but not of other proteins. In addition, using the new protocol we specifically labelled 14-3-3 proteins, protein disulfide isomerase and two unknown proteins of 45 and 46kDa in the mitochondrial-synaptosomal fraction of porcine cerebral cortex, none of which could be tagged by the previously used methods. The new methodology, which can be used for any sPLA2, constitutes a novel approach to discovering and purifying sPLA2-binding proteins, to studying the topology of their respective complexes and to following sPLA2s in different biological systems.
Lidija Kovacic; Jernej Sribar; Igor Krizaj
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2007-02-15
Journal Detail:
Title:  Bioorganic chemistry     Volume:  35     ISSN:  0045-2068     ISO Abbreviation:  Bioorg. Chem.     Publication Date:  2007 Aug 
Date Detail:
Created Date:  2007-07-03     Completed Date:  2007-09-05     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  1303703     Medline TA:  Bioorg Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  295-305     Citation Subset:  IM    
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
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MeSH Terms
Cross-Linking Reagents
Molecular Probe Techniques*
Phospholipases A / analysis,  metabolism*,  secretion
Phospholipases A2
Protein Binding
Protein Interaction Mapping / methods*
Viper Venoms / diagnostic use*
Reg. No./Substance:
0/Cross-Linking Reagents; 0/Viper Venoms; 58-85-5/Biotin; 97089-72-0/ammodytoxin A; EC 3.1.1.-/Phospholipases A; EC A2

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