A new photoprobe for studying biological activities of secreted phospholipases A2.
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.
Kovacic
Lidija
L
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Sribar
Jernej
J
Krizaj
Igor
I
D000818:Animals; D001710:Biotin; D003432:Cross-Linking Reagents; D006801:Humans; D015336:Molecular Probe Techniques; D010741:Phospholipases A / Q000032:analysis / Q000378:metabolism*; D054467:Phospholipases A2; D010777:Photochemistry; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D014757:Viper Venoms
D003432:Cross-Linking Reagents; D014757:Viper Venoms; D001710:Biotin; C068117:ammodytoxin A; D010741:Phospholipases A; D054467:Phospholipases A2