Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR.
(17/927)
Solid state 31P NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The 31P chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group. Macroscopically oriented MP-lipid samples between glass plates were studied by 31P NMR as a function of tilt angle. These spectra showed the coexistence of orientation-dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non-lamellar phases in DMPC/DMPG membranes. (+info)
Fluorescence, absorption and electron spin resonance study of bacteriochlorin a incorporation into membrane models.
(18/927)
Analysis of the bacteriochlorin a absorption spectra suggests the existence of a monomer-dimer equilibrium, particularly intense in phosphate buffer and favored by a decrease of the pH. The dye in methanolic solution is predominantly in monomeric form. Fluorescence and electron spin resonance nitroxide spin labeling measurements indicate that incorporation into the lipid phase of dimyristoyl-L-alpha-phosphatidylcholine liposomes induces dye monomerization. Moreover, the molecules are bound in the external surface of the vesicles and a complete incorporation is ensured by a lipid-to-dye ratio greater than 125. (+info)
Kinetic and equilibrium studies of incorporation of di-sulfonated aluminum phthalocyanine into unilamellar vesicles.
(19/927)
The interactions of cis-di-sulfonated aluminum phthalocyanine (PcS(2)Al) with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles have been investigated by fluorescence spectroscopy. At pH 7.0, PcS(2)Al incorporates into the vesicles with a high affinity constant (2.7x10(6) M(-1), in terms of phospholipid concentration). The fluorescence changes following rapid mixing of PcS(2)Al with vesicles are biphasic. The first phase is attributed to the entry of PcS(2)Al into the vesicles, as deduced from the linear dependence of the rate upon lipid concentration. More surprisingly, this rate is strongly pH dependent with a marked maximum around pH 7.3, a result interpreted in terms of the coordination state of the aluminum ion in aqueous solutions. At this pH, a hydroxide ion neutralizes the residual positive charge of the metal ion that remains unbalanced after coordination by the phthalocyanine cycle. A water molecule is likely to complete the metal coordination sphere. Only this form, PcAl(+)(OH(-))(OH(2)), with an uncharged core is quickly incorporated into the vesicles. The protonation of OH(-) or the deprotonation of the coordinated H(2)O leading to a positively or negatively charged core, respectively, account for the observed pH effect. Studies on the effect of cholesterol addition and exchange of PcS(2)Al between vesicles and albumin all indicate the absence of transfer of the phthalocyanine between the vesicle hemileaflets, a result expected from the presence of the two negatively charged sulfonated groups at the ring periphery. Instead, the slower kinetic phase is likely due to the movement of the phthalocyanine becoming more buried within the outer leaflet upon the loss of the water molecule coordinated to the aluminum ion. (+info)
Insect immune activation by recombinant Galleria mellonella apolipophorin III(1).
(20/927)
Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae. (+info)
Polymer-cushioned bilayers. I. A structural study of various preparation methods using neutron reflectometry.
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This neutron reflectometry study evaluates the structures resulting from different methods of preparing polymer-cushioned lipid bilayers. Four different techniques to deposit a dimyristoylphosphatidylcholine (DMPC) bilayer onto a polyethylenimine (PEI)-coated quartz substrate were examined: 1) vesicle adsorption onto a previously dried polymer layer; 2) vesicle adsorption onto a bare substrate, followed by polymer adsorption; and 3, 4) Langmuir-Blodgett vertical deposition of a lipid monolayer spread over a polymer-containing subphase to form a polymer-supported lipid monolayer, followed by formation of the outer lipid monolayer by either 3) horizontal deposition of the lipid monolayer or 4) vesicle adsorption. We show that the initial conditions of the polymer layer are a critical factor for the successful formation of our desired structure, i.e., a continuous bilayer atop a hydrated PEI layer. Our desired structure was found for all methods investigated except the horizontal deposition. The interaction forces between these polymer-supported bilayers are investigated in a separate paper (Wong, J. Y., C. K. Park, M. Seitz, and J. Israelachvili. 1999. Biophys. J. 77:1458-1468), which indicate that the presence of the polymer cushion significantly alters the interaction potential. These polymer-supported bilayers could serve as model systems for the study of transmembrane proteins under conditions more closely mimicking real cellular membrane environments. (+info)
Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.
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We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions. (+info)
Interaction of phloretin with lipid monolayers: relationship between structural changes and dipole potential change.
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Phloretin is known to adsorb to lipid surfaces and alters the dipole potential of lipid monolayers and bilayers. Its adsorption to biological and artificial membranes results in a change of the membrane permeability for a variety of charged and neutral compounds. In this respect phloretin represents a model substance to study the effect of dipole potentials on membrane permeability. In this investigation we studied the interaction of phloretin with monolayers formed of different lipids in the liquid-expanded and the condensed state. Phloretin integrated into the monolayers as a function of the aqueous concentration of its neutral form, indicated by an increase of the surface pressure in the presence of phloretin. Simultaneous recording of the surface potential of the monolayers allowed us to correlate the degree of phloretin integration and the phloretin-induced dipole potential change. Increasing the surface pressure decreased the phloretin-induced shift of the isotherms, but did not influence the phloretin-induced surface potential change. This means that phloretin adsorption to the lipid surface can occur without affecting the lipid packing. The surface potential effect of phloretin is accompanied by a change of the lipid dipole moment vector dependent on the lipid packing. This means that the relation between the surface potential change and the lipid packing cannot be described by a static model alone. Taking into account the deviations of the surface potential change versus molecular area isotherms of the experimental data to the theoretically predicted course, we propose a model that relates the area change to the dipole moment in a dynamic manner. By using this model the experimental data can be described much better than with a static model. (+info)
Condensed complexes of cholesterol and phospholipids.
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Mixtures of dihydrocholesterol and phospholipids form immiscible liquids in monolayer membranes at the air-water interface under specified conditions of temperature and 2-dimensional pressure. In recent work it has been discovered that a number of these mixtures exhibit two upper miscibility critical points. Pairs of upper critical points can be accounted for by a theoretical model that implies the cooperative formation of molecular complexes of dihydrocholesterol and phospholipid molecules. These complexes are calculated to be present in the membranes both above and below the critical points. Below the critical points the complexes form a separate phase, whereas above the critical points the complexes are completely miscible with the other lipid components. The cooperativity of complex formation prompts the use of the terminology condensed complex. (+info)