title: A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles.
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 associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies emphasize the importance of lateral packing of the lipids in C. atrox sPLA2 enzymatic hydrolysis of a membrane surface.
authors.affiliation: Department of Physics, Laboratory for Fluorescence Dynamics, University of Illinois at Urbana Champaign, Urbana, Illinois 61801, USA.
authors.forename: Susana A
authors.initials: SA
authors.lastname: Sanchez
authors.forename: Luis A
authors.initials: LA
authors.lastname: Bagatolli
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Theodore L
authors.initials: TL
authors.lastname: Hazlett
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D003435:Crotalid Venoms / Q000737:chemistry*; D017839:Crotalus / Q000378:metabolism*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D007848:Laurates / Q000494:pharmacology; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D017785:Photons; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
chemical_list: D003435:Crotalid Venoms; D005456:Fluorescent Dyes; D007848:Laurates; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C002448:1,2-linoleoylphosphatidylcholine; D015081:2-Naphthylamine; D010741:Phospholipases A; D054467:Phospholipases A2; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; D004134:Dimyristoylphosphatidylcholine; C065580:laurdan; C018525:1,2-dimyristoylphosphatidylethanolamine