A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles

Biophysical Journal
Susana A SánchezTheodore L Hazlett

Abstract

We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to assoc...Continue Reading

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Citations

Mar 26, 2010·The Journal of Membrane Biology·María S JaureguiberryOmar J Rimoldi
Apr 25, 2012·Proceedings of the National Academy of Sciences of the United States of America·Susana A SanchezEnrico Gratton
Dec 21, 2011·Journal of Lipid Research·Martín M Dodes TraianValeria Levi
Jan 30, 2014·Acta Biochimica Et Biophysica Sinica·María S JaureguiberryOmar J Rimoldi
Sep 25, 2010·Wiley Interdisciplinary Reviews. Systems Biology and Medicine·Senthil ArumugamPetra Schwille
Dec 9, 2008·Biochimica Et Biophysica Acta·Galya StanevaKamen Koumanov
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Jan 14, 2010·Biochimica Et Biophysica Acta·Maria Laura FananiRafael Gustavo Oliveira
Feb 1, 2008·Biophysical Journal·Adam Cohen Simonsen
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