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Susana A Sanchez

Publications and source records attributed to Susana A Sanchez.

5 recordsLinked to original sources
PubMed2005

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers.

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.

Apolipoprotein A-I↗
PubMed2002

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

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.

2-Naphthylamine↗
PubMed2002

Interaction of apolipoprotein A-I in three different conformations with palmitoyl oleoyl phosphatidylcholine vesicles.

Interactions of apolipoprotein A-I (apoA-I) with cell membranes appear to be important in the initial steps of reverse cholesterol transport. The objective of this work was to examine the effect of three distinct conformations of apoA-I (lipid-free and in 78 A or 96 A reconstituted high density lipoproteins, rHDL) on its ability to bind to, and abstract lipids from, palmitoyl oleoyl phosphatidylcholine membrane vesicles (small unilamellar vesicles, SUV, and giant unilamellar vesicles, GUV). The molecular interactions were observed by two-photon fluorescence microscopy, and the binding parameters were quantified by gel-permeation chromatography or isothermal titration microcalorimetry. Rearrangement of apoA-I-containing particles after exposure to SUVs was examined by native gel electrophoresis. The results indicate that lipid-free apoA-I binds reversibly, with high affinity, to the vesicles but does not abstract a significant amount of lipid nor perturb the vesicle structure. The 96 A rHDL, where all the amphipathic helices of apoA-I are saturated with lipid within the particles, do not bind to vesicles or perturb their structure. In contrast, the 78 A rHDL have a region of apoA-I, corresponding to a few amphipathic helical segments, which is available for external or internal phospholipid binding. These particles bind to vesicles with measurable affinity (lower than lipid-free apoA-I), abstract lipids from the membranes, and form particles of larger diameters, including 96 A rHDL. We conclude that the conformation of apoA-I regulates its binding affinity for phospholipid membranes and its ability to abstract lipids from the membranes.

Apolipoprotein A-I↗