Speciated High-Density Lipoprotein Biogenesis and Functionality.

Plasma high-density lipoprotein cholesterol (HDL-C) concentration is a negative risk factor for atherosclerotic cardiovascular disease (CVD). Despite this, most attempts to raise plasma HDL-C concentrations in a cardioprotective way have failed. Recently, hypotheses about the atheroprotective effects of HDL have shifted away from quantity to quality, mostly HDL function in reverse cholesterol transport. Plasma HDL from CVD patients is a poorer acceptor of cellular cholesterol than plasma from healthy controls, independent of plasma HDL-C concentrations. The function of HDL is likely determined by two other factors, stability and composition. The kinetic instability of HDL, which varies according to subclass, is a likely determinant of its reactivity in response to many HDL-modifying activities. HDL composition is also heterogeneous and variable; all HDL particles contain apo AI but only about two-thirds contain apo AII. This occurs despite the fact that apo AI and apo AII are hepatically secreted on separate HDL that later fuse in plasma. HDL also contains traces of other proteins, some of which have not yet been associated with HDL function. One minor HDL species are those that are secreted with intact signal peptides, which enhances their binding to HDL; these HDL have special properties that are independent of cholesterol transport. Here, we review and provide a perspective about what is currently known about speciated HDL biogenesis in the context of health and disease.

Plasma triglycerides determine low density lipoprotein composition, physical properties, and cell-specific binding in cultured cells.

The relationship between the plasma triglycerides and the LDL triglycerides of 30 normal and 48 hypertriglyceridemic subjects has been quantified; the data fit a simple adsorption isotherm, LDL triglyceride/(LDL triglyceride+LDL cholesterol ester) = 0.65 plasma triglyceride/(464 + plasma triglyceride). In vitro transfer of triglyceride from concentrated VLDL to VLDL-depleted plasma produced triglyceride-rich LDL that had similar properties. LDL uptake by HepG2 cells increased with LDL triglyceride content whereas the reverse was found with skin fibroblasts. At 37 degrees C, the cores of both normal and hypertriglyceridemic LDL were isotropic liquids. Circular dichroic spectra revealed no difference in the secondary structure of normal and triglyceride-rich LDL. The affinity of monoclonal antibody MB47, which binds to the receptor ligand of apo B-100 was independent of LDL triglyceride content. MB3, which binds near residue 1022 of apo B-100, showed a triglyceride-dependent decrease in affinity for LDL from hypertriglyceridemic subjects and from in vitro incubations. LDL with an elevated triglyceride content formed in vitro had reduced proteolytic cleavage of apo B-100 by Staphylococcus aureus V8 protease. From these data, we infer that (a) LDL triglyceride is a predictable function of plasma triglyceride, (b) triglyceride induces subtle changes in apo B-100 structure at a site that is remote from the putative receptor binding ligand, and (c) the triglyceride-dependent receptor-binding determinants of apo B-100 are recognized differently by fibroblasts and HepG2 cells.

In vivo interaction of synthetic acylated apopeptides with high density lipoproteins in rat.

The metabolism of synthetic peptide analogues of high density lipoprotein (HDL) apoproteins has been studied in the rat. These compounds are 15-amino acid lipid associating peptides (LAPs) bearing acyl chains of various lengths (0-16 carbon units). After injection of each 125I-LAP, the serum decay curves suggested a two-compartment process with a clearance rate decreasing when the acyl chain lengths increased. The similarity between the apparent half-life of C16-LAP and that of apoprotein A-I as well as the chromatographic analysis of rat serum were consistent with a partitioning of the LAPs between HDL and the aqueous phase. This was strongly dependent upon the acyl chain length of the LAPs. The distribution volumes of the 125I-LAPs in organs were measured 10 min after injection. The results were analyzed using a model explicitly predicting the organ distribution volumes of HDL and the equilibrium constant (Keq) of the binding of each LAP to HDL. HDL distributed significantly in the adrenals (250 microliters/g), liver (80 microliters/g), and ovaries (55 microliters/g), but not in the kidneys. This suggests that the binding of HDL apoproteins to kidneys, reported by others, was due to the uptake of free apoproteins. The Keqs exhibited a log-linear relationship with respect to the acyl chain length of the LAPs. Each carbon unit added to the acyl chain decreased the free energy of association by a constant value (0.3 kcal mol-1). This clearly showed a strict hydrophobic effect similar to that previously observed in vitro.

Increased sphingomyelin content of plasma lipoproteins in apolipoprotein E knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase.

Apolipoprotein E knockout (apoE0) mice accumulate atherogenic remnant lipoproteins in plasma. We now provide evidence that these particles are enriched in sphingomyelin (SM), and explore the mechanisms and possible pathophysiological consequences of this finding. The phosphatidylcholine/sphingomyelin (PC/SM) ratio was reduced in all lipoproteins in apoE0 mice compared with wild-type (Wt) mice (2.0+/-0.2 vs. 4.7+/-0.5; 2.8+/-0.5 vs. 5.5+/-0.9; 1.9+/-0. 5 vs. 4.6+/-0.5 for VLDL, LDL, and HDL), reflecting 400 and 179% increases in plasma pools of SM and PC, respectively. Turnover studies using [14C]PC/[3H]SM VLDL or HDL showed that the fractional catabolic rate (FCR) of VLDL-SM and HDL-SM were markedly reduced in the apoE0 mice compared with Wt mice, while the FCRs of VLDL-PC and HDL-PC were similar. By contrast, the FCRs of [3H]PC ether and [14C]SM were identical in apoE0 and Wt mice. The production rates of VLDL-SM and HDL-SM in apoE0 mice were much higher than in Wt mice, while the production rates of lipoprotein PC were similar. To assess the underlying mechanisms, we also measured the PC/SM ratio in VLDL and LDL of LDL receptor knockout (LDLr0) and hepatic LDL receptor-related protein knockout/LDLr0 mice, but found no difference with Wt mice. Using S-sphingomyelinase, an enzyme secreted by macrophages and endothelial cells, we found that VLDL and LDL from apoE0, but not from Wt or LDLr0 mice, were significantly aggregated, and that aggregation was not prevented by adding back apoE. We then enriched the apoE0-VLDL and Wt-VLDL with different amounts of SM, and found that VLDL aggregation was enhanced. Thus, the increased SM content of lipoproteins in apoE0 mice is due to combined synthesis and clearance defects. Impaired SM clearance reflects resistance to intravascular enzymes and delayed removal by a non-LDLr, non-LDLr related protein pathway. The increased SM content in slowly cleared remnant lipoproteins may enhance their susceptibility to arterial wall SMase and increase their atherogenic potential.

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length.

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)

Spectroscopic studies of the tyrosine residues of human plasma apolipoprotein A-II.

Human apolipoprotein A-II (apo A-II) in solution and associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was investigated by a combination of absorbance and fluorescence methods. Each apo A-II polypeptide chain contains four tyrosine residues but no tryptophan residues. Two and three tyrosine residues, respectively, appear to be buried for apo A-II in aqueous solution and in the lipid-associated protein. The spectroscopic properties of the tyrosine residues of lipid-associated apo A-II were also investigated. Plots of fluorescence intensity against temperature revealed a discontinuity in the region of the phase transition; however, over the same temperature range, there was no change in the exposure of tyrosine residues to the aqueous environment or in their mobility as measured by fluorescence polarization. Near-ultraviolet circular dichroic measurements demonstrated that the environments of the tyrosine residues of lipid-associated apo A-II and nitrated apo A-II were different from that of the apo A-II in solution or in a denatured state. Similar measurements also revealed that the microenvironments around tyrosines of apo A-II bound to DMPC in the gel phase are different from those observed in the liquid crystalline phase. Using environmentally sensitive fluorescence lipid probes, we have previously demonstrated that the polarity of the lipid/water interface of DMPC changes through a phase transition. The observations presented here indicate that these environmental changes also occur at the lipid/protein interface.

Effects of high-density lipoprotein(2) on cholesterol transport and acyl-coenzyme A:cholesterol acyltransferase activity in P388D1 macrophages.

High-density lipoproteins are the putative vehicles for cholesterol removal from monocyte-derived macrophages, which are an important cell type in all stages of atherosclerosis. The role of HDL(2), an HDL subclass that accounts for most variation in plasma HDL-cholesterol concentration, in cholesterol metabolism in monocyte-derived macrophages is not known. In this study, the dose-dependent effects of HDL(2) on cellular cholesterol mass, efflux, and esterification, and on cellular cholesteryl ester (CE) hydrolysis using the mouse macrophage P388D1 cell line was investigated. HDL(2) at low concentrations (40 microg protein/ml) decreased CE content without affecting cellular free cholesterol content (FC), CE hydrolysis, or cholesterol biosynthesis. In addition, HDL(2) at low concentrations reduced cellular acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity and increased FC efflux from macrophages. Thus, HDL(2) has two potential roles in reverse cholesterol transport. In one, HDL(2) is an acceptor of macrophage FC. In the other, more novel role, HDL(2) increases the availability of macrophage FC through the inhibition of ACAT. Elucidation of the mechanism by which HDL(2) inhibits ACAT could identify new therapeutic targets that enhance the transfer of cholesterol from macrophages to the liver.

Reversible folding reactions of human apolipoprotein A-I: pressure and guanidinium chloride effects.

Apolipoprotein A-I, the major structural polypeptide of human high-density lipoproteins, activates lecithin: cholesterol acyltransferase, the cholesterol ester-forming enzyme in plasma. Apolipoprotein A-I, like several other apolipoproteins, exhibits structural adaptability, which is manifest in a low free energy of stabilization and facile changes in secondary structure. We have investigated the dual effects of guanidinium chloride (GdmCl) and pressure perturbation at low GdmCl concentrations on apolipoproteins A-I conformational states, using fluorescence detection. Pressure alone (up to 3 kilobar) is insufficient to fully denature apolipoprotein A-I, and results in formation of metastable state(s). However, in conjunction with low concentrations of GdmCl the calculated volume change upon pressure denaturation increases from approx. -50 ml/mol to -90 ml/mol. The free energy of denaturation by pressure perturbation ranges from 1.4 to 1.8 kcal/mol, but the conformational states induced by pressure and GdmCl perturbation are most likely different. The physico-chemical properties of native and pressure-denatured conformational states can be, readily and reversibly, measured by fluorescence techniques. Biological activity of apolipoprotein A-I in the form of lecithin: cholesterol acyltransferase activation, is also reversible upon pressure perturbation. Samples of apolipoprotein A-I exposed to 2 kbar for an hour activated lecithin: cholesterol acyltransferase equally well as controls. To delineate more precisely the conformational states of apolipoprotein A-I under pressure, time-dependent anisotropy decay measurements, capable of resolving rotational heterogeneity, will be required.

Isolation and specificity of rat lecithin: cholesterol acyltransferase: comparison with the human enzyme using reassembled high-density lipoproteins containing ether analogs of phosphatidylcholine.

Rat plasma lecithin: cholesterol acyltransferase, a 68 kDa glycoprotein, has been purified 14 000-fold by a modification of a procedure used for the human enzyme. The activity of lecithin: cholesteryl acyltransferase in human and rat plasma are the same, although activation of both enzymes by human apolipoprotein A-I is greater than that produced by rat apolipoprotein A-I. Using reassembled high-density lipoproteins composed of human apolipoprotein A-I, phosphatidylcholine ethers and a series of different phosphatidylcholines, the separate effects of molecular species specificity and microenvironment on the rate of cholesteryl ester formation was determined. Substitution of a fluid lipid, 1-palmityl-2-oleyl-sn-glycero-3-phosphorylcholine, for a solid lipid, 1,2-dipalmityl-sn-glycero-3-phosphorylcholine, produced an 8-fold increase in the activity of all molecular species of phosphatidylcholine. With either solid or fluid lipid environments, the activity decreased as a function of increasing chain length of saturated acyl groups. Addition of one or more double bonds greatly increased the activity of a given saturated homologue. One major difference between the molecular specificity of rat and human lecithin: cholesteryl acyltransferase was that the latter had a two-fold preference for phosphatidylcholines containing arachidonate at the sn-2-position.

Plasma factors affecting the in vitro conversion of high-density lipoproteins labeled with a non-transferable marker.

We studied the in vitro conversion of HDL3 labeled with a radioiodinated diacyl lipid associating peptide (diLAP). DiLAP was previously shown to be nontransferable, which permitted its' use as a reliable marker of HDL particles. DiLAP-labeled HDL3 was incubated for 23 h at 37 degrees C in human or rat plasma or in reconstituted media containing delipidated plasma and/or lipoproteins and/or partially purified CETP. At the end of the incubations, the samples were adjusted to a density of 1.125 g/ml and ultracentrifuged. The two resulting fractions containing HDL2 and HDL3, respectively, were analyzed by gradient gel electrophoresis. Depending upon experimental conditions, diLAP-labeled HDL3 was converted into HDL2b- and/or small HDL3c-like particles. LCAT inhibition and to a lesser extent CETP promoted the formation of small HDL3c. Reactivation of LCAT led to the disappearance of small HDL3c. No HDL3c formed from HDL2 even in the absence of LCAT activity. When the incubations were performed in the presence of 100 mM thimerosal, which inhibited PLTP but not CETP activity, the conversion of diLAP-labeled HDL3 into HDL2 was almost completely blocked. Collective consideration of these data indicates that the formation of small HDL is moderately facilitated by CETP; that small HDL are converted to larger HDL species by LCAT and that the transformation of HDL3 into HDL2 is a process which largely depends upon PLTP activity.

Interaction of apoliprotein C-III with phosphatidylcholine vesicles. Dependence of aproprotein-phospholipid complex formation on vesicle structure.

We have studied the interaction of an apolipoprotein from human very low density lipoproteins (apoC-III) with egg yolk phosphatidylcholine in the form of single- and multi-bilayer vesicles. The reactivity of single-bilayer vesicles with apoC-III appears to be greater than that of the multi-bilayer vesicles according to several thermodynamic and spectrosconic criteria. In the complexes formed by the association of apoC-III with single-bilayer vesicles, the alpha-helical content of the peptide backbone and the apolarity of the environment around the tryptophan residues are greater than that observed in the complexes formed with the multibilayer vesicles. A higher yield and more homogeneous density distribution of lipid-apoprotein complexes results from the interaction of apoC-III with the single-bilayer vesicles relative to those obtained with the multi-bilayer vesicles. The enthalpy of association of apoC-III with phospholipid was greater for the single-shelled vesicles (25 kcal/mol apoC-III) than for the multi-shelled ones (18 kcal/mol apoC-III). The difference in reactivity of these two types of liposomes is not due to a difference in their fluidities since their fatty acid compositions are identical, but may be due to a difference in their areas of sterically accessible phospholipid, their permeabilities to the apoprotein, their radii of curvation, or a combination of these factors.

Thermodynamics of lipid-protein association. Enthalphy of association of apolipoprotein A-II with dimyristoylphosphatidylcholine-cholesterol mixtures.

Apolipoprotein A-II spontaneously associates with dimyristoylphosphatidylcholine (DMPC)-cholesterol mixtures to give products whose composition is a sensitive function of temperature and cholesterol content. At most temperatures, the lipid-to-protein stoichiometry of the product recombinant increases with increasing mol% cholesterol. Up to about 18 mol% cholesterol, the complexes have the same average sterol/DMPC ratio as that of the starting mixtures. At 24 mol% cholesterol or higher, no detectable lipid/protein complex formed. At 37 degrees C, the lipid-to-protein stoichiometry is essentially constant, irrespective of the cholesterol content and substitution of unsaturated phospholipids for DMPC. The enthalpy of lipid-protein association is a function of cholesterol content and, at 25 degrees C, increases linearly with the mol% cholesterol in the reaction mixture until it becomes endothermic between 15 and 20 mol% cholesterol. The results fit a model in which cholesterol is excluded from phospholipids in the 'boundary' layer, which is perturbed by the protein. At high cholesterol concentrations, the formation of a recombinant is thermodynamically unfavorable.

Kinetics of spontaneous and plasma-stimulated sphingomyelin transfer.

The mechanism of transfer of a pyrene-labeled sphingomyelin (PySM) between different lipid compartments was studied by a fluorescence technique. The first-order kinetics are independent of donor and acceptor concentration and the identity of the acceptor; the rates are accelerated by 'structure-breaking' solutes and inhibited by 'structure-making' solutes. These observations are consistent with the transfer of PySM occurring via the aqueous phase that separates the donor and acceptor compartments. We have partially purified a plasma factor that stimulates the transfer rate. Our in vitro results suggest that both spontaneous and stimulated transfer might contribute to the redistribution of sphingomyelin in vivo,

Kinetics of tryptic hydrolysis as a probe of the structure of human plasma apolipoprotein A-II.

As a model system to understand apolipoprotein structure-function and their relationships to proteolytic events, the kinetics of tryptic hydrolysis of apolipoprotein A-II (apo A-II) was investigated in solution and in association with phospholipid. The rates of appearance and identities of specific peptides were determined by reversed-phase high-performance liquid chromatography and amino acid analysis, respectively. For the kinetics of hydrolysis of apo A-II in solution, the carboxyl-terminal peptides of residues 55-77 and 56-77 appeared first, followed by peptides of residues 4-23, 29-39, 40-44 and 45-54, which appeared at nearly identical rates. The kinetics of hydrolysis of apo A-II associated with 1,2-dimyristoyl-sn-glycero-3-phosphocholine showed several differences. First, a 100-fold larger amount of trypsin was needed to obtain a similar rate of product formation; second, a new peptide appeared, eluting earlier than apo A-II but having a similar amino acid composition; and third, the relative rates of appearance of peptides were different. The secondary structure surrounding the bonds susceptible to trypsin cleavage was determined by several predictive algorithms. The lysine amino acid bonds were found to be in regions defined by a high helical amphipathic moment. The reduced susceptibility to tryptic hydrolysis of apo-II associated with phospholipid appears to be due to a higher free energy of stabilization of protein secondary structure. As a consequence, the lysine amino acid bonds are in folded regions of the protein where they are conformationally inaccessible to enzymatic hydrolysis. By use of structure-prediction methods, it is possible to designate which regions of apolipoproteins may be important in proteolysis.

Action of lecithin:cholesterol acyltransferase on model lipoproteins. Preparation and characterization of model nascent high density lipoprotein.

Apolipoprotein A-I, the major protein of human plasma high density lipoprotein, is the primary activator of plasma lecithin:cholesterol acyltransferase. In vitro, the association of apolipoprotein A-I with physiological phosphatidylcholines can be catalyzed by mixing the protein and lipid with sodium cholate, which is removed by chromatography. The apolipoprotein A-I/phospholipid complex has the physical properties of an HDL, and when cholesterol is present the complex is a highly reactive substrate in the lecithin:cholesterol acyltransferase-catalyzed reaction. The relative reactivity of this complex compared with a number of other lipid-protein complexes is presented and discussed.

Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine.

The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.

A pH titration study on the ionic bridging within lipopolysaccharide aggregates.

The packing of lipopolysaccharide aggregates from rough strains of Escherichia coli was examined at different pH values. Lipopolysaccharide head-group motion, measured with an electron spin resonance probe, was found to be dependent on pH, and indicated the existence of multiple ionizable groups. Lipopolysaccharide from a rough (Ra) and a heptose-less (Re) mutant were more rigid at pH 5 than at pH 10.5. In addition, head-group mobility of the magnesium salt of Ra lipopolysaccharide was substantially less than that of the sodium salt at pH 7.0, whereas at high pH (pH 12) the two salts were equally fluid. Changes in head-group packing were also reflected in pH-dependent changes in the phase transition measured with differential scanning calorimetry. The enthalpy of the transition, delta Ht, for the sodium salt of Re lipopolysaccharide was greatest at pH 7.5 and approached zero in both the acidic and the basic pH ranges. We propose that fixed charges in the core and lipid A regions significantly influence lipopolysaccharide head-group motion and the lipopolysaccharide aggregation state. Furthermore, ionic bridging among phosphate groups dramatically rigidifies head group interactions in the neutral to acidic pH ranges.

Fluorescence assay of the specificity of human plasma and bovine liver phospholipid transfer proteins.

The specificities of a human plasma and bovine liver phospholipid transfer protein were studied using a fluorescence assay based on the transfer of pyrenyl phospholipids. This method was used previously to determine the mechanism of spontaneous transfer of phospholipids between model lipoproteins (Massey, J.B., Gotto, A.M., Jr. and Pownall, H.J. (1982) Biochemistry 21, 3630-3636). The pyrenyl phospholipids varied in the headgroup moiety; pyrenyl phosphatidylcholines contained different fatty acyl chains in the sn-1 position. Model high-density lipoproteins (R-HDL) consisting of apolipoprotein A-I and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were used as donor and acceptor particles. As previously shown, the bovine liver protein mediated the transfer of only phosphatidylcholine. In contrast, the human plasma protein transferred all species studied which included a phosphatidylserine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, sphingomyelin, galactosylcerebroside, and a diacylglycerol. The activity of these transfer proteins was only slightly affected by changes in the acyl chain composition of the transferring lipid. Pyrenyl and radioactive ([3H]POPC) phospholipids were transferred with equal rates by the human transfer protein, suggesting that this protein has similar binding characteristics for pyrenyl and natural phospholipids. Spontaneous phospholipid transfer occurs by the aqueous diffusion of monomeric lipid where the rate is highly dependent on fatty acyl chain composition. In this study, no correlation between the rate of spontaneous transfer and protein-mediated transfer was found. The apparent Km values for R-HDL and low-density lipoprotein (LDL), when used as acceptors, were similar when based on the number of acceptor particles. The apparent Vmax for the bovine liver protein was identical for R-HDL and LDL but for the plasma protein Vmax was slightly higher for R-HDL. These results suggest that, like the bovine liver protein, the plasma protein functions as a phospholipid-binding carrier that exchanges phospholipids between membrane surfaces. The assay of lipid transfer proteins by pyrenyl-labeled lipids is faster and easier to perform than other current methods, which require separation of donor and acceptor particles, and is suitable for studies on the function and mechanism of action of lipid transfer proteins.

The properties of membranes formed from cyclopentanoid analogues of phosphatidylcholine.

We have examined the thermal characteristics and barrier properties of vesicles formed from six analogues of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). These analogues differ from DPPC in that the glycerol backbone has been replaced by each of the diastereoisomeric cyclopentane-1,2,3-triols. All of these compounds have main gel to liquid-crystal phase transition temperatures within 5 Kelvin of DPPC and four possess comparable enthalpies and entropies of transition. For two of the analogous, however, the values of the enthalpy and entropy of transition are more than double that of DPPC. The permeability characteristics and organization (as measured by diphenylhexatriene fluorescence depolarization) of vesicles formed from these two compounds suggest that their large transition enthalpy and entropy result from either a reorganization of the polar head group region during the transition or interdigitation of the acyl chains of opposing monolayers.

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)nonanoic 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 C18 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)nonanoyl] 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.