Search

The effect of phosphatidylserine and sterol carrier proteins on cholesterol exchange was determined using an assay not requiring separation of donor and acceptor membrane vesicles. Sterol carrier protein-2 (SCP, also called nonspecific lipid transfer protein), but not fatty acid binding protein (FABP, also called sterol carrier protein), enhanced the initial rate of sterol exchange between neutral zwitterionic phosphatidylcholine small unilamellar vesicles (SUV) 2.3-fold. Phosphatidylserine at 10 mol% increased the initial rate of spontaneous and of SCP-mediated (but not FABP-mediated) sterol exchange by 22% and 44-fold, respectively. The SCP potentiation of sterol transfer was dependent on SCP concentration and on phosphatidylserine concentration. The SCP-mediated sterol transfer was inhibited by a variety of cations including KCl, divalent metal ions, and neomycin. The data suggest that SCP increase in activity for sterol transfer may be partly ascribed to charge on the phospholipid. [ABSTRACT FROM AUTHOR]

Dendrimers and other cationic macromolecules in general are considered potential vehicles for intracellular drug delivery. We used fluorescence spectroscopy to study the interaction of cationic PAMAM (polyamidoamine) dendrimers with model lipid bilayers. When the dendrimers interacted with small unilamellar vesicles (SUV) loaded with the self-quenching fluorophore calcein they did not induce release of calcein neither with neutral SUV (made of egg phosphatidylcholine) nor negatively charged SUV (with 25% egg phosphatidylglycerol). The G1 (generation 1, Mr = 1430) dendrimer was labeled with fluorescein and the G4 dendrimer (Mr = 14215) with a similar fluorophore Oregon green, and their membrane interactions were followed by changes in fluorescence anisotropy during titrations with lipid. The anisotropy changed linearly with the density of negative charge in the membrane. The addition of high salt caused redissociation of the dendrimers from SUV. These two observations prove the electrostatic nature of the interaction. Analysis of the binding data yielded values of the dissociation constants Kd. For G1-dendrimer, Kd = 30 +/- 16 uM with neutral SUV and Kd = 11 +/- 3 M with negatively charged SUV. For G4, which carries much higher charge on its surface, exhibited lower dissociation constants, Kd = 16 +/- 7 uM with neutral SUV and 5 +/- 1 uM with negatively charged SUV. We conclude that the interaction of PAMAM dendrimers with lipid membrane is electrostatic and the dendrimer binding does not significantly disrupt the bilayer integrity. [ABSTRACT FROM AUTHOR]

Cyt1A is a cytolytic toxin produced by the spore-forming bacterium Bacillus thuringiensis var. israelensis, used in insecticide preparations. Understanding the molecular details of how the toxin changes conformation in the presence of lipid membrane is important for elucidating the toxin's mode of action. Previous binding studies were performed using membranes made of chemically undefined lipid egg phosphatidylcholine (PC). Here we studied lipid specificity of Cyt1A binding regarding saturation of fatty acyl chain and chemical nature of the lipid head-group. Fluorescence of tryptophan was used as a measure of binding. We measured Cyt1A binding to membranes made of the saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) at 7°C and 37°C and determined the apparent dissociation constant (Kd) to be 111 ± 211 µM and 19.7 ± 5.9 µM, respectively. The apparent Kd of Cyt1A binding to membranes made of the egg PC lipid mixture was previously determined to be 91 µM. Thus at lower temperature, Cyt1A binds to membranes made of DMPC to a similar extent as to those made of the egg PC lipid mixture, but binds more strongly to DMPC at higher temperature. This suggests that Cyt1A binds to vesicles composed of either saturated or unsaturated lipids, and it binds saturated lipids in the liquid-crystalline state more strongly than unsaturated lipids. [ABSTRACT FROM AUTHOR]

The mechanism of internalization of cationic peptides into the cells is unknown. We used fluorescence spectroscopy to study the interaction between Protein Transduction Domain of the HIV-1 TAT protein (TAT-PTD; residues 47-60 of TAT, fluorescently labeled with tryptophan) and the lipid bilayer in the form of small unilamellar lipid vesicles (SUV). The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and increase in anisotropy upon the interaction with SUV, which was proportional with the negative charge density in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. Kd1 was independent of the negative charge density and accounted for 24% of the interaction, whereas Kd2, contributing 76% to the interaction, decreased linearly with the density of negative charge in the membrane, suggesting an electrostatic nature of the latter interaction. The former could not be inhibited by high salt, suggesting its van der Waals or hydrophobic nature. TAT-PTD did not dissipate membrane potential (165 mV, negative inside), nor did it affect fluorescence anisotropy of the membrane fluorescence probes TMA-DPH or DPH, indicating preserved membrane integrity upon TAT-PTD binding. When the pyrene-labeled phosphatidyl glycerol was included in the membrane, TAT-PTD induced pyrene excimer formation, but only at the temperature above the melting point of the lipid. Thus, the two prerequisites for a strong binding of TAT-PTD to the lipid membrane are the presence of the negative charge and the liquid crystalline phase of the lipid bilayer. [ABSTRACT FROM AUTHOR]