No abstract

The mechanism of cholesterol and phosphatidylcholine exchange has been investigated by following the transfer of radiolabeled cholesterol and phosphatidylcholine from negatively charged, unilamellar cholesterol-egg yolk phosphatidylcholine donor vesicles to neutral acceptor vesicles of similar composition. Vesicles were incubated in the absence of protein and were stable to fusion over the course of the experiment. At intervals, donor and acceptor vesicles were separated by passage through a column of DEAE-Sepharose; less than 1% of the charged and 80-95% of the neutral vesicles were recovered in the eluate. Over 12 h at 37 degrees C, 90% of the donor vesicle [4-14C]cholesterol was transferred to the acceptor vesicles in a first-order process whose half-time was 2.3 +/- 0.3 h. This indicates that transfer of cholesterol molecules from the inner to outer monolayer of the vesicle bilayer is not rate limiting in exchange. In contrast to cholesterol exchange, the half-time for 1-palmitoyl-2-oleoyl[1-14C]phosphatidylcholine exchange was 48 +/- 5 h so that more than six molecules of cholesterol were transferred for each molecule of phosphatidylcholine. The interfacial flux of cholesterol from the donor bilayer is 5.3 x 10(-15) mol cm-2 s-1 (approximately 3 molecules/min for an average vesicle) and is similar to fluxes observed in other systems where phosphatidylcholine or cholesterol ester exchange is catalyzed by an exchange protein. When the acceptor vesicle concentration was increased 20-fold in cholesterol exchange experiments or 9-fold in phosphatidylcholine exchange experiments, the rate of label transfer was not affected. The activation energy of cholesterol exchange between 15 and 37 degrees C was 73 +/- 5 kJ mol-1. Transfer of cholesterol across a dialysis membrane is shown to be a slow process whose rate may be predicted by application of Fick's first law of diffusion. These results are only consistent with a mechanism of lipid exchange in which cholesterol and phosphatidylcholine diffuse through the aqueous phase; the experimental activation energy is associated with desorption of lipid from the donor bilayer into the aqueous phase.

Human apolipoprotein A-I (apo A-I) possesses multiple tandem repeating 22-mer amphipathic alpha-helixes. Computer analysis and studies of model synthetic peptides and recombinant protein-lipid complexes of phospholipids have suggested that apo A-I interacts with HDL surface lipids through cooperation among its individual amphipathic helical domains. To delineate the overall lipid-associating properties of apo A-I, the first step is to understand the lipid-associating properties of individual amphipathic helical domains. To this end, we synthesized and studied each of the eight tandem repeating 22-mer domains of apo A-I: residues 44-65, 66-87, 99-120, 121-142, 143-164, 165-186, 187-208, and 220-241. Among the 22-mers, only the N- and C-terminal peptides (44-65 and 220-241) were effective in clarifying multilamellar vesicles (MLVs) of dimyristoylphosphatidylcholine (DMPC). These two peptides also exhibited the highest partition coefficient into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine liposomes, the highest exclusion pressure for penetration into an egg yolk phosphatidylcholine monolayer, and the greatest reduction in the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. These results suggest that the strong, lipid-associating properties of apo A-I are localized to the N- and C-terminal amphipathic domains. Although each of the eight peptides studied has an amphipathic structure, models based on changes in residual effective amino acid hydrophobicity resulting from differing depths of helix penetration into the lipid are best able to explain the high lipid affinity possessed by the two terminal domains. Differential scanning calorimetry (DSC) studies showed that on a molar basis, apo A-I is about 10 times more effective than the most effective peptide analyzed in reducing the enthalpy of the gel-to-liquid crystalline phase transition of DMPC MLVs. Because previous proteolysis experiments coupled with the present DSC results suggest that the lipid-associating domains of apo A-I are distributed throughout the length of the 243 amino acid residues, we propose that the terminal amphipathic helical domains are involved in the initial binding of apo A-I to the lipid surface to form HDL particles, followed by cooperative binding of the middle six amphipathic helical domains, perhaps aided by salt-bridge formation between adjacent helixes arranged in an antiparallel orientation.

The rates of [14C]cholesterol transfer from small unilamellar vesicles containing cholesterol dissolved in bilayers of different phospholipids have been determined to examine the influence of phospholipid-cholesterol interactions on the rate of cholesterol desorption from the lipid-water interface. The phospholipids included unsaturated phosphatidylcholines (PC's) (egg PC, dioleoyl-PC, and soybean PC), saturated PC (dimyristoyl-PC and dipalmitoyl-PC), and sphingomyelins (SM's) (egg SM, bovine brain SM, and N-palmitoyl-SM). At 37 degrees C, for vesicles containing 10 mol% cholesterol, the half-times for exchange are about 1, 13, and 80 h, respectively, for unsaturated PC, saturated PC, and SM. In order to probe how differences in molecular packing in the bilayers cause the rate constants for cholesterol desorption to be in the order unsaturated PC greater than saturated PC greater than SM, nuclear magnetic resonance (NMR) and monolayer methods were used to evaluate the cholesterol physical state and interactions with phospholipid. The NMR relaxation parameters for [4-13C]cholesterol reveal no differences in molecular dynamics in the above bilayers. Surface pressure (pi)-molecular area isotherms for mixed monolayers of cholesterol and the above phospholipids reveal that SM lateral packing density is greater than that of the PC with the same acyl chain saturation and length (e.g., at pi = 5 mN/m, where both monolayers are in the same physical state, dipalmitoyl-PC and palmitoyl-SM occupy 87 and 81 A2/molecule, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of exchange of phosphatidylcholine and lysophosphatidylcholine from unilamellar donor vesicles to acceptor vesicles of similar composition were followed in a protein-free system to establish the relationship between the rate of exchange and the aqueous-phase solubility of the lipid. Further, the rate of exchange of dimyristoylphosphatidylcholine (DMPC) between vesicles was examined over a range of temperatures to determine the effect of the lipid phase transition on the rate of lipid exchange. Intervesicular exchange of DMPC is faster than transbilayer exchange; lipid molecules in the outer monolayer of the bilayer exchange with t1/2 = 2.0 h at 37 degrees C. A discontinuity is observed in Arrhenius plots of DMPC exchange; the activation energy over the temperature range 27-45 degrees C is 70 kJ mol-1. The t1/2 for DMPC exchange extrapolated to 24.5 degrees C (the phase transition temperature of the donor bilayer) is 6.5 h and from temperatures below 24 degrees C is 82.6 h. The differences in the thermodynamic parameters of activation for DMPC exchange above and below 24.5 degrees C are 25 kJ mol-1 for the activation enthalpy and 197 J mol-1 K-1 for the activation entropy. These differences are similar to the enthalpy and entropy changes associated with the gel to liquid-crystalline phase transition of DMPC. The rate of exchange of lysopalmitoyl-phosphatidylcholine (LPPC) was difficult to measure since LPPC transfers rapidly to the columns used for separating donor and acceptor vesicles; the t1/2 for transfer is less than 2 min. LPPC at 5 mol % in cholesterol-egg PC vesicles does not affect the rate of cholesterol exchange.(ABSTRACT TRUNCATED AT 250 WORDS)