Spontaneous Phospholipid Transfer Research Articles

Spontaneous phospholipid transfer: development of a quantitative model

The effects of lipid structure on the kinetics of spontaneous transfer of a series of phosphatidylcholines have been determined. Donors, which were model-reassembled high-density lipoproteins composed of apo A-I, 1-palmitoyl-2-oleoylphosphatidylcholine, and a trace of a radiolabeled lipid, were mixed with acceptors, which were human low-density lipoproteins. Within a series of phosphatidylcholines, the addition of double bonds and methylene units, respectively, increased and decreased the rate of transfer in a predictable way. An equation that predicts the rates of transfer of a large number of diacylglycerides and phosphoglycerides from any lipoprotein has been empirically derived from these data. The transfers of phosphatidylcholines that contain superpolyunsaturated fatty acids (four or more double bonds) do not obey the derived equation, probably due to limitations on the number of conformational degrees of freedom in these lipids. The range of measured transfer halftimes extends from less than 2 h to more than 12 days. Thus, the spontaneous transfer halftimes of some (but not all) lipids are short compared to the lifetime of lipoproteins in plasma. These results suggest that some lipids transfer among lipoproteins and cells via a spontaneous mechanism while others require specific transfer factors or hydrolysis to achieve this within a physiologically significant time frame.

Mechanism of spontaneous, concentration-dependent phospholipid transfer between bilayers

We have previously demonstrated that spontaneous phospholipid transfer between bilayer vesicles at higher vesicle concentrations is characterized not only by a first-order desorption rate but also by a second-order process dependent on vesicle concentration (Jones & Thompson, 1989b). We have extended our studies to examine the mechanism of this second-order process by investigating transfer as a function of lipid type, temperature, aqueous medium composition, and vesicle size. The results suggest a mechanism of concentration-dependent transfer in which the rate of lipid monomer desorption from vesicle bilayers is enhanced in transient vesicle-vesicle complexes.

Lipid transfers between reconstituted high density lipoprotein complexes and low density lipoproteins: effects of plasma protein factors

In this study we examined the transfer of lipids between reconstituted high density lipoprotein discs (r-HDL) and human low density lipoproteins (LDL) in the presence and absence of lecithin:cholesterol acyltransferase (LCAT) or of plasma phospholipid transfer protein (PLTP). We found that spontaneous transfer of phospholipids from r-HDL to LDL occurred by an apparent first order reaction with a half-time of 5.8 to 6.9 hr depending on the phospholipid. During the time of incubation of r-HDL with LDL (from 0 to 25 hr), the phospholipid content of r-HDL decreased more than 30%, the free cholesterol content increased 2.5-fold, and low levels of cholesteryl esters appeared in r-HDL. These compositional changes gave rise to small discoidal particles with a limiting diameter of 77 A and two molecules of apoA-I per particle. When LCAT was included in the reaction mixture, the r-HDL lost even more phospholipid, lost some free cholesterol, and gained cholesteryl esters relative to the apolipoprotein content, due to the enzymatic reaction. The products of the LCAT reaction had a diameter of 93 A and three, rather than two, apoA-I molecules per particle. Inclusion of PLTP into the reaction mixture accelerated the transfer of phospholipids (half-time of 1.7 hr) and the formation of the 77 A product. In addition to these compositional and morphological changes, which may be important in the interconversions of native HDL subspecies, the prolonged incubations revealed some slow reactions, such as the esterification of LDL cholesterol by LCAT, a background formation of cholesteryl esters in r-HDL, and an apparent hydrolysis of cholesteryl esters in LDL in the presence of r-HDL.

Low concentrations of bile salts increase the rate of spontaneous phospholipid transfer between vesicles.

The rate of 1-palmitoyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino] dodecanoyl] phosphatidylcholine (P-C12-NBD-PC) transfer between dioleoylphosphatidylcholine vesicles was measured by a technique based on resonance energy transfer between P-C12-NBD-PC and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726]. Addition of bile salts at concentrations below their critical micelle concentrations increased the rate of spontaneous P-C12-NBD-PC transfer without disrupting the vesicles. The effectiveness in increasing the transfer rate was dependent on the structure of the bile salt. In general, conjugated bile salts were more effective than unconjugated, and mono- and dihydroxy bile salts were more effective than trihydroxy. The kinetics of intervesicular P-C12-NBD-PC transfer in the presence of cholate were found to be consistent with a mass action kinetic model based on the premise that bile salts bind to the vesicles, alter the dissociation and/or association rate constants for phospholipid monomer-vesicle interaction, and increase the rate of phospholipid transfer via the diffusion of soluble monomers through the aqueous phase. Temperature dependence studies indicated that cholate binding to vesicles is an entropy-driven process and that cholate binding lowers the free energy of activation for phospholipid monomer-vesicle dissociation by producing compensatory decreases in both the enthalpy and entropy of activation.

Nonspecific lipid transfer protein (sterol carrier protein 2) is bound to rat liver mitochondria: its role in spontaneous intermembrane phospholipid transfer

In the present study we have investigated the transfer of phospholipids between vesicles and rat liver mitochondria. Transfer was measured by electron paramagnetic resonance spectroscopy using vesicles that contained spin-labeled phospholipids. A spontaneous transfer was observed which could be strongly inhibited by treating the mitochondria with the thiol reagent mersalyl. Transfer was also greatly reduced after a saline wash of the mitochondria; the transfer activity was then recovered in the wash. This activity was inhibited by tryptic digestion and mersalyl. By gel chromatography, enzyme immunoassay and immunoblotting it was demonstrated that the activity in the wash was due to the nonspecific lipid transfer protein (sterol carrier protein 2). We could estimate that up to 85% of the spontaneous phospholipid transfer between vesicles and rat liver mitochondria was mediated by this transfer protein.

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid Chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)non-anoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [ 3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C 18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)non-anoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.

Kinetics and mechanism of the spontaneous transfer of fluorescent phospholipids between apolipoprotein-phospholipid recombinants. Effect of the polar headgroup.

Fluorescent derivatives of a phosphatidylglycerol, phosphatidylserine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, and diacylglycerol have been studied to establish the effect of different polar headgroups on the mechanism and kinetics of spontaneous phospholipid transfer between recombinants of human plasma apolipoprotein A-II and dimyristoylphosphatidylcholine. The fluorescent lipids are all 1-myristoyl-2-[9-(1-pyrenyl)nonanoyl] glycerides. The transfer of the lipids is a first order process where the rate is independent of the concentration over a 50 fold range of the acceptor recombinants. These results are consistent with the lipids transferring as monomers being a water-soluble intermediate. The rate of transfer of the different phospholipids are slightly slower than phosphatidylcholine, with that of phosphatidylethanolamine being about 4 times slower. The transfer of phospholipids with a titratable headgroup is pH-dependent. The difference in the rates and pH dependence may be a function of the interactions (hydrogen bonding) between polar headgroups. The rate of transfer of the diacylglycerol is 20 times slower than phosphatidylcholine, but its activation energy (21 kcal/mol) is only 2 to 3 kcal less than most of the phospholipids (23 kcal/mol). These results suggest that the rate and activation energy for the spontaneous transfer of phospholipids can be predicted to a first approximation on the basis of its hydrophobic content, irrespective of the pH or identity of the polar headgroup.

Corrections - Mechanism of the Spontaneous Transfer of Phospholipids between Bilayers

Corrections - Mechanism of the Spontaneous Transfer of Phospholipids between Bilayers

Spontaneous phospholipid transfer between artificial vesicles followed by free-flow electrophoresis

The application of continuous free-flow electrophoresis in the liposome field is described. The technique is able to distinguish between artificial phospholipid vesicles as a function of their surface charge. This made possible to follow intervesicular interactions between differently charged bilayer structures. As an example, we present evidence for a relatively fast, spontaneous phospholipid transfer between dimyristoylphosphatidylcholine and mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol vescles.

Mechanism of the spontaneous transfer of phospholipids between bilayers.

A fluorescent phospholipid, 1-palmitoyl-2-pyrenedecanoylphosphatidylcholine, was used to study the mechanism of spontaneous phospholipid transfer between single-walled phospholipid vesicles. The half-time for transfer of this molecule between vesicles of dimyristoylphosphatidylcholine at 36 degrees C is 13 h if flip-flop is negligible or 24 h if flip-flop is faster than intervesicle exchange. The half-time is unaffected by the concentration of acceptor vesicles, which indicates that transfer of label takes place by diffusion of monomers or micelles through the aqueous phase rather than by collision of vesicles. These results are compared with previous studies of spontaneous lipid transfer.

Complete exchange of phospholipids between microsomes and plasma lipoproteins mediated by liver phospholipid-exchange proteins

It is known that cholesterol and phospholipids are in dynamic equilibrium and spontaneously exchange both in vitro and in vivo between individual classes of plasma lipoproteins [l-4] . Spontaneous transfer of phospholipids has also been shown between lipoproteins and erythrocytes [5],mitochondria [6] ,liver microsomes [7] , liver slices [8] and cells in culture [9]. In these examples of phospholipid exchange it is presumed that transfer occurs upon formation of a collision complex. Evidence for a protein-facilitated transfer of lipoprotein phospholipids in serum is controversial [7,10]. On the other hand, eukaryotic cells contain proteins designated as phospholipid exchange proteins (PLEP) that do facilitate the transfer of phospholipids from one membrane to another [ 1 I-131 . A purified PLEP from beef liver shows a highly specific affinity for phosphatidylcholine (PC-PLEP [ 141). Two proteins have been isolated from bovine cerebral cortex which stimulate both the transfer of phosphatidylinositol (PI) and PC (PI-PLEP [IS] ). Recently, exchange proteins have been isolated from rat liver [ 161 and hepatoma [ 171 which lack specificity in the transfer of phospholipid. The purpose of the present study was to determine whether and if so, to what extent, purified PCand PI-exchange proteins catalyze the transfer of phospholipids between rat liver microsomes and the individual classes of lipoproteins. It will be shown that the protein-mediated transfer of labeled PC and PI from microsomes to very low density lipoproteins (VLDL), LDL and HDL was 5-lo-fold higher than