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P-C4-15 Lipid specificity in α-actinin binding to lipid membranes
C4 Lipid-protein interactions and dynamics P-C4-13 CALORIMETRIC DETECTION OF PROTON INDUCED FUSION OF LIPID VESICLES WENK M, SEELIG J Department of Biophysics, Biocenter, Univ.of Base1(CH)
P-c4-14 THE EFFECT OF TFWWWMBRANE POTENTIAL ON THE STRUCTURE OF LIPID BILAYER MALINSKY J., HERMAN P., VECER J., PLASEK J. Institute of Physics, Charles University, Prague (CZ)
Purpose: Fusion of lipid membranesis a process of outmost importance in living cells which up to date lacksthorough description on a physicalchemicalbasis.Here we followed fusion of lipid vesiclesby meansof a calorimetric techniqueand determined the enthalpy of fusion.
Purpose: The value of transmembrane potential in living cells is typically -1OOmV. Does such an electric field modify the structure of a phospholipid bilayer? Methods: Unilamellar DMPC liposomes were prepared, stained with DPH. The different values of the transmembrane potential were set. These vesicles were studied by time-resolved fluorescenceanisotropy.
Methods: Reaction heats were measuredusing isothermal titration calorimetry. Results: Lysophosphatidylcholine (C16:0-LPC) inhibits membranefusion. In the absenceof LPC, vesiclescomposedof 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine(POPE)and oleic acid were induced to fuse by a drop in pH. In presenceof LPC fusion is inhibited. The difference of the reaction heats then yields a fusion enthalpy of AH = 400 - 600 calmor’ of total lipid.
Results: In vesicleswith non-zero transmembrane potential changes in limitting anisotropy rinrhave been found. Temperature-induced phase transitions in lipid bilayer are shown to be sensitiveto both Nernstian and surface potentials. Comparable experimets with TMA-DPH are included.
Conclusions: Isothermal titration calorimetry, delivering a direct physicalquantity, is a suitable method to follow model membrane fusion. Deconvolution of the overall enthalpy of fusion into its different components and dependencies requiresfurther investigations.
Conclusions: The structure of pure lipid bilayer is modified by transmembrane potential. The magnitude of the electric field required for these changesis comparable with physiologic values. P-C4-16 INTERACTION BETWEEN NYSTATIN AND PHOSPIWLIPID VESICLES: THE STEROL DEPENDENCE. COUTlNHO A, PRlETO M. CQFM,ComplexoI, I.S.T.,Lisboa(Portugal)
P-W-15 LIPID SPECIFICITY IN a-ACTININ BINDING TO LIPID MEMBRANES GANG LI, X. HAN, G.-H. LI, K.-C. LlN Biophysics,Beijing Medical University (CN) Purpose: a-Actinin hasbeen postulated to be a linker protein between actin filaments and the membrane. But it remainsto be clarified how aactinin binds to the membrane, and this has been the subjectof the present study.
Purpose: It is well known that ergosterol-rich fungal membranesare much more sensitiveto the action of the polyene antibiotics than cholesterolrich membranes, characteristic of mammalian cells. However, the underlying mechanism that explainsthis difference is still controversial. Methods: The interaction between Nystatin and cholesterol- and ergosteroltontaining LUVs of POPC(with 0, 5, 10, 20 and 30 mol% of sterol) was studied both by steady-state and timeiresolvedfluorescencemeasurements. Results: Nystatin has an higher partition coefficient for ergosterol- than for cholesterolcontaining lipid vesicles. Moreover, both the fluorescence spectra of the antibiotic and its mean fluorescence lifetime were formd to be concentration-dependent only in ergosterolcontaining LUVs. Conclusions: Nystatin readily self-associates only in ergosterolcontftining vesicles. Our results suggestthat the role of sterols in Nystatin mode of action is not mediated through a ehabge of the physicalproperties of the bilayer.
Methods: Binding of a-actinin to large unilamellar lipid vesicleswas investigated by use of resonanceenergy transfer and proteolysis. Actin bundles formed on lipid monolayers of various lipid compositions were examined by EM methods. Results: a-Actinin binds to membranes which contain anionic phospholipids.Actin bundles formed on lipid monolayersonly when anionic phospholipid, DG and PApresent together in the lipid monolayer. Conclusions: Anionic phospholipid is essential for the binding of a-actinin to membrane. DG and PA are not essentialfor the bind, but required for the bundling of actin filaments by a-actinin
in the membranous environment.
120
P-c4-14 THE EFFECT OF TFWWWMBRANE POTENTIAL ON THE STRUCTURE OF LIPID BILAYER MALINSKY J., HERMAN P., VECER J., PLASEK J. Institute of Physics, Charles University, Prague (CZ)
Purpose: Fusion of lipid membranesis a process of outmost importance in living cells which up to date lacksthorough description on a physicalchemicalbasis.Here we followed fusion of lipid vesiclesby meansof a calorimetric techniqueand determined the enthalpy of fusion.
Purpose: The value of transmembrane potential in living cells is typically -1OOmV. Does such an electric field modify the structure of a phospholipid bilayer? Methods: Unilamellar DMPC liposomes were prepared, stained with DPH. The different values of the transmembrane potential were set. These vesicles were studied by time-resolved fluorescenceanisotropy.
Methods: Reaction heats were measuredusing isothermal titration calorimetry. Results: Lysophosphatidylcholine (C16:0-LPC) inhibits membranefusion. In the absenceof LPC, vesiclescomposedof 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine(POPE)and oleic acid were induced to fuse by a drop in pH. In presenceof LPC fusion is inhibited. The difference of the reaction heats then yields a fusion enthalpy of AH = 400 - 600 calmor’ of total lipid.
Results: In vesicleswith non-zero transmembrane potential changes in limitting anisotropy rinrhave been found. Temperature-induced phase transitions in lipid bilayer are shown to be sensitiveto both Nernstian and surface potentials. Comparable experimets with TMA-DPH are included.
Conclusions: Isothermal titration calorimetry, delivering a direct physicalquantity, is a suitable method to follow model membrane fusion. Deconvolution of the overall enthalpy of fusion into its different components and dependencies requiresfurther investigations.
Conclusions: The structure of pure lipid bilayer is modified by transmembrane potential. The magnitude of the electric field required for these changesis comparable with physiologic values. P-C4-16 INTERACTION BETWEEN NYSTATIN AND PHOSPIWLIPID VESICLES: THE STEROL DEPENDENCE. COUTlNHO A, PRlETO M. CQFM,ComplexoI, I.S.T.,Lisboa(Portugal)
P-W-15 LIPID SPECIFICITY IN a-ACTININ BINDING TO LIPID MEMBRANES GANG LI, X. HAN, G.-H. LI, K.-C. LlN Biophysics,Beijing Medical University (CN) Purpose: a-Actinin hasbeen postulated to be a linker protein between actin filaments and the membrane. But it remainsto be clarified how aactinin binds to the membrane, and this has been the subjectof the present study.
Purpose: It is well known that ergosterol-rich fungal membranesare much more sensitiveto the action of the polyene antibiotics than cholesterolrich membranes, characteristic of mammalian cells. However, the underlying mechanism that explainsthis difference is still controversial. Methods: The interaction between Nystatin and cholesterol- and ergosteroltontaining LUVs of POPC(with 0, 5, 10, 20 and 30 mol% of sterol) was studied both by steady-state and timeiresolvedfluorescencemeasurements. Results: Nystatin has an higher partition coefficient for ergosterol- than for cholesterolcontaining lipid vesicles. Moreover, both the fluorescence spectra of the antibiotic and its mean fluorescence lifetime were formd to be concentration-dependent only in ergosterolcontaining LUVs. Conclusions: Nystatin readily self-associates only in ergosterolcontftining vesicles. Our results suggestthat the role of sterols in Nystatin mode of action is not mediated through a ehabge of the physicalproperties of the bilayer.
Methods: Binding of a-actinin to large unilamellar lipid vesicleswas investigated by use of resonanceenergy transfer and proteolysis. Actin bundles formed on lipid monolayers of various lipid compositions were examined by EM methods. Results: a-Actinin binds to membranes which contain anionic phospholipids.Actin bundles formed on lipid monolayersonly when anionic phospholipid, DG and PApresent together in the lipid monolayer. Conclusions: Anionic phospholipid is essential for the binding of a-actinin to membrane. DG and PA are not essentialfor the bind, but required for the bundling of actin filaments by a-actinin
in the membranous environment.
120