Mechanism of the phospholipid transfer protein-mediated transfer of phospholipids from model lipid vesicles to high density lipoproteins

To study the effects of the phospholipid transfer protein (PLTP) on the thermodynamic parameters governing the transfer of phospholipids (PL) from single bilayer vesicles (SBV) to high density lipoprotein (HDL), we performed transfer measurements at various temperatures between 4 and 65 degrees C, using a pyrenylphosphatidylcholine (Pyr-PC) as probe. The proportion of excimer (E) to monomer (M) fluorescence of a pyrenyl moiety constitutes a direct measure of its local concentration. The transfers of Pyr-PC were monitored by following the decrease of E/M. The data were used to calculate the rate constants K(+1) for the transfer from SBV to HDL and to generate the corresponding Arrhenius plots. The equilibrium constants, K(eq), for the same reactions were also determined and used to generate Van't Hoff plots. From these data, we calculated the thermodynamic parameters for both the whole transfer reaction and the transition state. Both K(+1) and K(eq) values clearly varied with temperature. PLTP induced very similar decreases in the free energy for the whole reaction (DeltaG) and in that for the transition state (DeltaG(#)). At 37 degrees C, the decreases were of 0.37 and 0.29 kcal/mol, respectively. We studied the thermal denaturation of PLTP between 37 and 65 degrees C, and the effects of denatured PLTP samples on the PL transfer reaction were then determined. In all cases, the changes of DeltaG remained comparable to those of DeltaG(#). Thus the essential action of PLTP is to facilitate the first step of the reaction, which can be considered as the desorption of PL molecules from the surface of donor particles.     

Inflammatory platelet-activating factor-like phospholipids in oxidized low density lipoproteins are fragmented alkyl phosphatidylcholines.

Oxidation of human low density lipoprotein (LDL) generates proinflammatory mediators and underlies early events in atherogenesis. We identified mediators in oxidized LDL that induced an inflammatory reaction in vivo, and activated polymorphonuclear leukocytes and cells ectopically expressing human platelet-activating factor (PAF) receptors. Oxidation of a synthetic phosphatidylcholine showed that an sn-1 ether bond confers an 800-fold increase in potency. This suggests that rare ether-linked phospholipids in LDL are the likely source of PAF-like activity in oxidized LDL. Accordingly, treatment of oxidized LDL with phospholipase A(1) greatly reduced phospholipid mass, but did not decrease its PAF-like activity. Tandem mass spectrometry identified traces of PAF, and more abundant levels of 1-O-hexadecyl-2-(butanoyl or butenoyl)-sn-glycero-3-phosphocholines (C(4)-PAF analogs) in oxidized LDL that comigrated with PAF-like activity. Synthesis showed that either C(4)-PAF was just 10-fold less potent than PAF as a PAF receptor ligand and agonist. Quantitation by gas chromatography-mass spectrometry of pentafluorobenzoyl derivatives shows the C(4)-PAF analogs were 100-fold more abundant in oxidized LDL than PAF. Oxidation of synthetic alkyl arachidonoyl phosphatidylcholine generated these C(4)-PAFs in abundance. These results show that quite minor constituents of the LDL phosphatidylcholine pool are the exclusive precursors for PAF-like bioactivity in oxidized LDL.     

Interaction of locust apolipophorin III with lipoproteins and phospholipid vesicles: effect of glycosylation

Apolipophorin III (apoLp-III) from Locusta migratoria is an exchangeable apolipoprotein that binds reversibly to lipoprotein surfaces. The native protein is glycosylated at Asn-18 and Asn-85. Variable attachment of five distinct oligosaccharide moieties at the two glycosylation sites results in molecular weight heterogeneity, as seen by mass spectrometry. The main mass peak of 20,488 Da decreases to 17,583 Da after removal of carbohydrate, indicating that apoLp-III carbohydrate mass is approximately 14% by weight. Deglycosylated apoLp-III induced clearance of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol vesicles at a faster rate than glycosylated apoLp-III. However, in lipoprotein binding assays, in which apoLp-III interacts with surface-localized diacylglycerol, only minor differences in binding were observed. The fluorescence properties of 1-anilinonaphthalene-8-sulfonate were unaffected by the glycosylation state of apoLp-III, indicating that no changes in the relative amount of exposed hydrophobic surface occurred as a result of carbohydrate removal. We propose that glycosyl moieties affect the ability of apoLp-III to transform phospholipid bilayer vesicles into disc-like complexes by steric hindrance. This is due to the requirement that apoLp-III penetrate the bilayer substrate prior to conformational opening of the helix bundle. On the other hand, the glycosyl moieties do not affect lipoprotein binding interactions as it does not involve deep protein penetration into the lipid milieu. Rather, lipoprotein binding is based on oriented protein contact with the lipid surface followed by opening of the helix bundle, which allows formation of a stable interaction with surface exposed hydrophobic sites.     

Different types of interaction of oxidized and reduced cytochrome c with phospholipid vesicles]

The binding constants of ferri- and ferrocytochrome c interactions with phosphatidylcholine or cardiolipin-containing vesicules were determined. It was found that affinity of ferricytochrome c to phospholipids is one order of magnitude higher that of ferrocytochrome c. A comparative investigation of circular dichroism spectra of free and phospholipid-bound ferri- and ferrocytochrome c was undertaken and it was shown that alpha-helix content of free ferrocytochrome c is higher than that of ferricytochrome c. The formation of ferricytochrome c containing lipoprotein complex led to decrease of alpha-helix content of the protein. In the case of ferrocytochrome c on the other hand interaction with phospholipids did not cause any changes in alpha-helix content. Distribution of ferri- and ferrocytochrome c in different two-phase systems consisting of dextran and polyethylenglycol or dextran and polyethylenglvcol-palmitate was also studied. A comparison of distribution constants shows that higher alpha-helix content of ferrocytochrome c results in the formation of hydrophobic clusters in the protein molecules. In previous communications it was reported that binding of ferrocytochrome c to phospholipids is determined by hydrophobic interactions while in the case of ferricytochrome c the interactions with phospholipids are mainly electrostatic. On the basis of the results obtained in this work it is supposed that it is hydrophobic clusters which determine the binding of ferrocytochrome c to phospholipid membranes.     

Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis

In order to determine the effects of a plasma phospholipid transfer protein on the transfer of phospholipids from very low density lipoproteins (VLDL) to high density lipoproteins (HDL) during lipolysis, biosynthetically labeled rat 32P-labeled VLDL was incubated with human HDL3 and bovine milk lipoprotein lipase (LPL) in the presence of the plasma d greater than 1.21 g/ml fraction or a partially purified human plasma phospholipid transfer protein (PTP). The addition of either the PTP or the d greater than 1.21 g/ml fraction resulted in a 2- to 3-fold stimulation of the transfer of phospholipid radioactivity from VLDL into HDL during lipolysis. In the absence of LPL, the PTP caused a less marked stimulation of transfer of phospholipid radioactivity. Both the d greater than 1.21 g/ml fraction and the PTP enhanced the transfer of VLDL phospholipid mass into HDL, but the percentage transfer of phospholipid radioactivity was greater than that of phospholipid mass, suggesting stimulation of both transfer and exchange processes. Stimulation of phospholipid exchange was confirmed in experiments where PTP was found to augment transfer of [14C]phosphatidylcholine radioactivity from HDL to VLDL during lipolysis. In experiments performed with human VLDL and human HDL3, both the d greater than 1.21 g/ml fraction and the PTP were found to stimulate phospholipid mass transfer from VLDL into HDL during lipolysis. Analysis of HDL by non-denaturing polyacrylamide gradient gel electrophoresis showed that enhanced lipid transfer was associated with only a slight increase in particle size, suggesting incorporation of lipid by formation of new HDL particles. In conclusion, the plasma d greater than 1.21 g/ml fraction and a plasma PTP enhance the net transfer of VLDL phospholipids into HDL and also exchange of the phospholipids of VLDL and HDL. Both the transfer and exchange activities of PTP are stimulated by lipolysis.     

Mixing of oxidized and bilayer phospholipids

Composition and phase dependence of the mixing of 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), with the oxidized phospholipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) were investigated by characterizing the aggregation states of DPPC/PGPC and DOPC/PGPC using a fluorescence quenching assay, dynamic light scattering, and time-resolved fluorescence quenching in the temperature range 5–60 °C. PGPC forms 3.5 nm radii micelles of aggregation number 33. In the gel phase, DPPC and PGPC fuse to form mixed vesicles for PGPC molar fraction, X_PGPC ≤ 0.3 and coexisting vesicles and micelles at higher X_PGPC. Data suggest that liquid phase DPPC at 50 °C forms mixed vesicles with segregated or hemi fused DPPC and PGPC for X_PGPC ≤ 0.3. At 60 °C, DPPC and PGPC do not mix, but form coexisting vesicles and micelles. DOPC and PGPC do not mix in any proportion in the liquid phase. Two dissimilar aggregates of the sizes of vesicles and PGPC micelles were observed for all X_PGPC for T ≥ 22 °C. DOPC–PGPC and DPPC–PGPC mixing is non-ideal for X_PGPC > 0.3 in both gel and fluid phases resulting in exclusion of PGPC from the bilayer. Formation of mixed vesicles is favored in the gel phase but not in the liquid phase for X_PGPC ≤ 0.3. For X_PGPC ≤ 0.3, aggregation states change progressively from mixed vesicles in the gel phase to component segregated mixed vesicles in the liquid phase close to the chain melting transition temperature to separated coexisting vesicles and micelles at higher temperatures.     

Reconstitution of Saccharomyces cerevisiae phosphatidylserine synthase into phospholipid vesicles. Modulation of activity by phospholipids

Membrane-associated phosphatidylserine synthase was purified from Saccharomyces cerevisiae (Bae-Lee, M., and Carman, G. M. (1984) J. Biol. Chem. 259, 10857-10862) and reconstituted into phospholipid vesicles containing phosphatidylcholine/phosphatidylethanolamine/ phosphatidylinositol/phosphatidylserine. Reconstitution was performed by removing detergent from an octyl glucoside/phospholipid/Triton X-100/enzyme mixed micelle by Sephadex G-50 super-fine chromatography. The average diameter of the vesicles was 90 nm, and the enzyme was reconstituted asymmetrically with the active site facing outward. The enzymological properties of reconstituted phosphatidylserine synthase were determined in the absence of detergent. The enzyme was reconstituted into vesicles with phospholipid compositions approximating those of wild type and mutant strains of S. cerevisiae. Reconstituted activity was modulated by the phosphatidylinositol/phosphatidylserine ratio in the vesicles. The modulation of activity observed in the vesicles is enough to account for some of the fluctuations in the phosphatidylserine content in vivo.     

Exchange of phospholipids between unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and plasma very low density lipoproteins.

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange [14C]dipalmitoyl phosphatidylcholine from sonicated vesicles to human plasma very low density lipoproteins (VLDL). The exchange of [14C]-dipalmitoyl phosphatidylcholine for VLDL phospholipids was temperature dependent and linear with respect to time and amount of exchange protein. In the absence of the exchange protein, less than 10% of the [14C]dipalmitoyl phosphatidylcholine was transferred. At an initial weight ratio of [14C]-dipalmitoyl phosphatidylcholine vesicles to VLDL phospholipid (1.2 mg) of 2.2, the exchange protein (14 microgram) replaced 55% of the VLDL phospholipids with [14C]dipalmitoyl phosphatidylcholine in 15 min; VLDL protein and cholesterol content were unaltered. From these studies we conclude that the exchange protein is a useful method to alter the phospholipid composition of VLDL under conditions such that there is minimal perturbation of the lipoprotein.     

Encapsulation of hemoglobin in phospholipid vesicles

Hemoglobin has been encapsulated in phospholipid vesicles by extrusion of hemoglobin/lipid mixtures through polycarbonate membranes. This technique avoids the use of organic solvents, sonication, and detergents which have proven deleterious to hemoglobin. The vesicles are homogeneous, with a mean size of 2400 A as determined by photon correlation spectroscopy. The encapsulated hemoglobin binds oxygen reversibly and the vesicles are impermeable to ionic compounds. Hemoglobin encapsulated in egg phosphatidylcholine vesicles converts to methemoglobin within 2 days at 4 degrees C. By contrast, when a mixture of dimyristoyl phosphatidylcholine, cholesterol and dicetyl phosphate is used there is no acceleration in methemoglobin formation, and the preparation is stable for at least 14 days at 4 degrees C.     

Mass spectrometry analysis of oxidized phospholipids

The evidence that oxidized phospholipids play a role in signaling, apoptotic events and in age-related diseases is responsible for the increasing interest for the study of this subject. Phospholipid changes induced by oxidative reactions yield a huge number of structurally different oxidation products which difficult their isolation and characterization. Mass spectrometry (MS), and tandem mass spectrometry (MS/MS) using the soft ionization methods (electrospray and matrix-assisted laser desorption ionization) is one of the finest approaches for the study of oxidized phospholipids. Product ions in tandem mass spectra of oxidized phospholipids, allow identifying changes in the fatty acyl chain and specific features such as presence of new functional groups in the molecule and their location along the fatty acyl chain. This review describes the work published on the use of mass spectrometry in identifying oxidized phospholipids from the different classes.     

Axon-myelin transfer of phospholipids and phospholipid precursors

Previous studies have provided evidence for axon-to-myelin transfer of intact lipids and lipid precursors for reutilization by myelin enzymes. Several of the lipid constituents of myelin showed significant contralateral/ipsilateral ratios of incorporated radioactivity, indicative of axonal origin, whereas proteins and certain other lipids did not participate in this transfer-reutilization process. The present study will examine the labeling of myelin phosphoinositides by this pathway. Both 32PO4 and [3H]inositol were injected monocularly into 7-9-wk-old rabbits and myelin was isolated 7 or 21 days later from pooled optic tracts and superior colliculi. In total lipids 32P counts of the isolated myelin samples showed significant contralateral/ipsilateral ratios as well as increasing magnitude of contralateral-ipsilateral differences during the time interval. Thin-layer chromatographic isolation of the myelin phosphoinositides revealed significant 32P-labeling of these species, with PIP and PIP2 showing time-related increases. This resembled the labeling pattern of the major phospholipids from rabbit optic system myelin in a previous study and suggested incorporation of axon-derived phosphate by myelin-associated enzymes. The 32P label in PI, on the other hand, remained constant between 7 and 21 days, suggesting transfer of intact lipid. This was supported by the labeling pattern with [3H]inositol, which also showed no increase over time for PI. These results suggest axon-myelin transfer of intact PI followed by myelin-localized incorporation of axon-derived phosphate groups into PIP and PIP2. The general topic of axon-myelin transfer of phospholipids and phospholipid precursors is reviewed.     

Water permeability of phospholipid vesicles

Summary The water permeability of phospholipid vesicles 0.5 to 10 in diameter bounded by one or by several lipid bilayers was measured by following the change in turbidity of a suspension after mixing in a stopped flow apparatus. A semi-empirical formulation for evaluating volume changes of vesicles with a broad size range by measurement of turbidity is developed. The rate of flow is analyzed in terms of reaction rate theory. The water permeability coefficients for phosphatidylcholine vesicles were approximately 44 /sec at 25°C and 70 /sec at 37°C. The activation energy for water transport was 8.25 kcal/mole. The results were consistent with the view that water permeates by dissolution and diffusion in the membrane.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

The insertion of D-beta-hydroxybutyrate apodehydrogenase into phospholipid monolayers and phospholipid vesicles

The strong interaction of D-beta-hydroxybutyrate dehydrogenase with phospholipid monomolecular films is demonstrated by the surface pressure increase of a film compressed up to 33 mN/m. Although the D-beta-hydroxybutyrate apodehydrogenase was able to penetrate many phospholipid monolayers, it interacted preferentially with negatively charged monolayers such as those made from diphosphatidylglycerol. The weakest interaction was found with phosphatidylcholine, which is the reactivating phospholipid for the enzyme. These interactions were dependent on the phospholipid chain length, ionic strength, and pH. At basic pH the apoenzyme lost its specificity for negatively charged phospholipids, suggesting the deprotonation of a cationic amino acid residue of the enzyme polypeptide chain. The charge effects are in agreement with results obtained using phospholipid vesicles. Beside the electrostatic interactions, the influence of phospholipid chain length and the ionic strength indicate that D-beta-hydroxybutyrate apodehydrogenase penetrates into the hydrophobic part of the lipid interface.     

Antibodies against phospholipids and oxidized LDL in alcoholic patients

Antiphospholipid antibodies (APA) are a generic term describing antibodies that recognize various phospholipids. Hepatocyte damage is a cardinal event in the course of alcoholic liver injury and autoantibodies against phospholipids could play an important role in this process. APA in alcoholic patients seem to reflect membrane lesions, impairment of immunological reactivity, liver disease progression and they correlate significantly with disease severity. LDL oxidation is supposed to be one of the most important pathogenic mechanisms of atherosclerosis and antibodies against oxidized low-density lipoprotein (oxLDL) are some kind of an epiphenomenon of this process. The scope of our study was to determine some autoantibodies (IgG-oxLDL and antiphospholipid antibodies) and their possible changes in alcoholic patients. We studied IgG-oxLDL and four APA - anticardiolipin antibodies (ACA), antiphosphatidylserine antibodies (APSA) antiphosphatidylethanolamine antibodies (APE) and antiphosphatidylcholine antibodies (APCA) in 35 alcoholic patients with mildly affected liver function at the beginning of the abuse treatment. The control group consisted of 60 healthy blood donors. In the studied group, we obtained positive results concerning total ACA in 17.1 % of alcoholic patients (8.3 % in the control group), 11.4 % IgG-ACA (6.7 %), 8.6 % IgM-ACA (3.3 %), 14.3 % total APE (6.7 %), 14.3 % total APCA (8.3 %) and 20 % total APSA (8.3 % in the control group). The IgG-oxLDL (406.4+/-52.5 vs 499.9+/-52.5 mU/ml) was not affected in alcoholic patients. We conclude that the autoantibodies against oxLDL are present in sera of alcoholics and healthy blood donors. Based on our results which revealed a wide range of IgG-oxLDL titres in the healthy population, this parameter does not appear to be very promising for the evaluation of the risk of atherosclerosis. Alcoholics with only mild affection of liver functions did not exhibit a significantly higher prevalence of all studied antiphospholipid antibodies (ACA, APSA, APE, APCA) which could lead to membrane lesions in these patients.