Phase behavior and glass transition of 1,2-dioleoylphosphatidylethanolamine (DOPE) dehydrated in the presence of sucrose

The effect of sucrose on the phase behavior of 1,2-dioleoylphosphatidylethanolamine (DOPE) as a function of hydration was studied using differential scanning calorimetry and X-ray diffraction. DOPE/sucrose/water dispersions were dehydrated at osmotic pressures (Pi) ranging from 2 to 300 MPa at 30 degrees C and 0 degrees C. The hexagonal II-to-lamellar gel (H(II)-->L(beta)) thermotropic phase transition was observed during cooling in mixtures dehydrated at Pior=57 MPa, the H(II)-->L(beta) thermotropic phase transition was precluded when sucrose entered the rigid glassy state while the lipid was in the H(II) phase. Sucrose also hindered the H(II)-to-lamellar crystalline (L(c)), and H(II)-to-inverted ribbon (P(delta)) lyotropic phase transitions, which occurred in pure DOPE. Although the L(c) phase was observed in dehydrated 2:1 (mole ratio) DOPE/sucrose mixtures, it did not form in mixtures with higher sucrose contents (1:1 and 1:2 mixtures). The impact of sucrose on formation of the ordered phases (i.e., the L(c), L(beta), and P(delta) phases) of DOPE was explained as a trapping of DOPE in a metastable H(II) phase due to increased viscosity of the sucrose matrix. In addition, a glass transition of DOPE in the H(II) phase was observed, which we believe is the first report of a glass transition in phospholipids.     

Isolation and characterization of the major apolipoprotein from chicken high density lipoproteins.

High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen high-density lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I. Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NH2-terminal sequence of Asp-Glu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of alpha-helical structures; the calculated helicity was 90%. Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.     

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis

HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation.

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Plasma lipoprotein changes attending the intravenous administration of Triton WR-1339 in normolipidemic dogs: preferential effect on high density lipoproteins

The nonionic detergent Triton WR-1339 was injected intravenously into normolipidemic dogs in a single dose of 150 mg/kg body weight followed by three other injections (75 mg/kg) on days 2, 6, and 12. The Triton produced a significant elevation of the plasma cholesterol of these animals, but not of their triglyceride levels, and profound changes of their plasma lipoproteins, particularly of the high density lipoprotein class. These changes were dependent on the concentration of Triton attained in plasma; when the levels were above 1.5 mg/ml, density gradient ultracentrifugation, electrophoretic, and chemical analyses indicated that an interaction between Triton and HDL had occurred. This interaction was attended by a gradual loss of the surface components of HDL, namely apoA-I, phospholipids, and unesterified cholesterol, and by the appearance of two cholesteryl ester-rich lipoproteins of d 1.019-1.024 g/ml and d 1.038-1.058 g/ml containing apoA-I and proteins with electrophoretic mobilities of apoB, apoE, and apoA-IV. At the time that these changes had occurred, the activities of the enzymes lecithin: cholesterol acyltransferase and post-heparin lipase were unaffected. When 125I-labeled apoA-I was injected intravenously into animals receiving Triton, the residence time of the radiolabeled protein in plasma increased from a control value of 3.1 days to 7.2 days. However, the apparent half-times of the radiolabeled apoA-I varied among the lipoprotein fractions it was associated with: d 1.119-1.159 g/ml, 5.28 days; d 1.019-1.024 g/ml, 7.55 days, and d 1.038-1.058 g/ml, 5.39 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Phospholipids bound with plasma proteins in children with vitamin D-deficient rickets

Composition of phospholipids bound with plasma proteins in the healthy children and in those with vitamin-D-deficient rickets are studied. It is found that the quantitative and qualitative content of phospholipids in the low- and high-density lipoproteins increases considerably with the rickets. At the same time the content of phospholipids which form complexes wit fibrinogen gets two times lower. The character of changes in the phospholipid composition in protein fractions and in the whole plasma in case of rickets is different.     

Lateral distribution of phospholipid and cholesterol in apolipoprotein A-I recombinants

The influence of cholesterol on the assembly and structure of model high-density lipoproteins (HDL) has been investigated. Model HDL composed of apolipoprotein A-I (apoA-I) and 1,2-dimyristoylphosphatidylcholine (DMPC) formed spontaneously at the transition temperature (Tc) of the lipid. Those composed of apoA-I and 1-palmitoyl-2-oleoylphosphatidylcholine were formed by a cholate dialysis method. At low cholesterol/phospholipid ratios both lipids and assembly methods yielded a model HDL whose composition was identical with that of the initial mixture; as the cholesterol/phospholipid ratio of the initial mixture was increased, the fraction of cholesterol appearing in the model HDL decreased, and a negative correlation between the cholesterol and protein contents of the model HDL was observed. At high cholesterol/phospholipid ratios the association of apoA-I and phospholipids appeared to be thermodynamically unfavorable. The effects of cholesterol content on the thermal properties of a model HDL composed of DMPC and apoA-I were further investigated by differential scanning calorimetry, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, fluorescence energy transfer, and excimer fluorescence of pyrenyl derivatives of phosphatidylcholine (PC) and cholesterol. The addition of cholesterol decreased the transition enthalpy of DMPC, raised the midpoint of the transition, and modulated motional freedom in the phospholipid matrix. The amount of cholesterol required to produce these effects was lower in the model HDL than in multilamellar liposomes. In a model HDL composed of DMPC and apoA-I, the lateral diffusion of a pyrene-labeled cholesterol was dramatically changed at the Tc whereas little change was observed in that of a pyrene-labeled PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and motion of phospholipids in human plasma lipoproteins. A 31P NMR study

The structure and motion of phospholipids in human plasma lipoproteins have been studied by using 31P NMR. Lateral diffusion coefficients, DT, obtained from the viscosity dependence of the 31P NMR line widths, were obtained for very low density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoproteins (HDL2, HDL3), and egg PC/TO microemulsions at 25 degrees C, for VLDL at 40 degrees C, and for LDL at 45 degrees C. At 25 degrees C, the rate of lateral diffusion in LDL (DT = 1.4 x 10(-9) cm2/s) is an order of magnitude slower than in the HDLs (DT = 2 x 10(-8) cm2/s). At 45 degrees C, DT for LDL increases to 1.1 x 10(-8) cm2/s. In contrast, DT for VLDL increases only slightly going from 25 to 40 degrees C. The large increase in diffusion rate observed in LDL occurs over the same temperature range as the smectic to disordered phase transition of the core cholesteryl esters, and provides evidence for direct interactions between the monolayer and core. In order to prove the orientation and/or order of the phospholipid head-group, estimates of the residual chemical shift anistropy, delta sigma, have been obtained for all the lipoproteins and the microemulsions from the viscosity and field dependence of the 31P NMR line widths. For VLDL and LDL, the anisotropy is 47-50 ppm at 25 degrees C, in agreement with data from phospholipid bilayers. For the HDLs, however, significantly larger values of 69-75 ppm (HDL2) and greater than 120 ppm (HDL3) were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calorimetric studies of cytochrome oxidase-phospholipid interactions

Thermotropic phase transitions in phospholipid vesicles reconstituted with mitochondrial cytochrome oxidase (EC 1.9.3.1) were studied using differential scanning calorimetry. Both dimyristoylphosphatidylcholine (DMPC) and mixtures of DMPC and cardiolipin were used at different lipid-to-protein ratios. The incorporated protein reduces the energy absorbed during phase transitions of DMPC vesicles, and causes a small decrease in the transition temperature (tm). delta H depends on the amount of protein in the vesicles. This dependence indicates that about 72 DMPC molecules are influenced per cytochrome alpha alpha 3 monomer. The transition parameters remain unaffected by changes in ionic strength or by reduction of the enzyme. Incorporation of cytochrome oxidase depleted of subunit III into DMPC liposomes resulted in a larger decrease of tm, but the amount of perturbed phospholipids remains similar to that in the case of the intact enzyme. Incorporation of cytochrome oxidase into DMPC/cardiolipin vesicles counteracts the effect of cardiolipin in decreasing the enthalpy of the DMPC transition. Thus cytochrome oxidase segregates the phospholipids by attracting cardiolipin from the bulk lipid. Cytochrome c does not significantly affect this apparent cardiolipin 'shell' around membranous cytochrome oxidase.     

Intracellular apoA-I and apoB distribution in rat intestine is altered by lipid feeding

Intracellular forms of chylomicrons, very low density lipoprotein (VLDL) and high density lipoprotein (HDL) have previously been isolated from the rat intestine. These intracellular particles are likely to be nascent precursors of secreted lipoproteins. To study the distribution of intracellular apolipoprotein among nascent lipoproteins, a method to isolate intracellular lipoproteins was developed and validated. The method consists of suspending isolated enterocytes in hypotonic buffer containing a lipase inhibitor, rupturing cell membranes by nitrogen cavitation, and isolating lipoproteins by sequential ultracentrifugation. ApoB and apoA-I mass are determined by radioimmunoassay and newly synthesized apolipoprotein characterized following [3H]leucine intraduodenal infusion. Intracellular chylomicron, VLDL, low density lipoprotein (LDL), and HDL fractions were isolated and found to contain apoB, and apoA-IV, and apoA-I. In the fasted animal, less than 10% of total intracellular apoB and apoA-I was bound to lipoproteins and 7% of apoB and 35% of apoA-I was contained in the d 1.21 g/ml infranatant. The remainder of intracellular apolipoprotein was in the pellets of centrifugation. Lipid feeding doubled the percentage of intracellular apoA-I bound to lipoproteins and increased the percentage of intracellular apoB bound to lipoproteins by 65%. Following lipid feeding, the most significant increase was in the chylomicron apoB and HDL apoA-I fractions. These data suggest that in the fasting state, 90% of intracellular apoB and apoA-I is not bound to lipoproteins. Lipid feeding shifts intracellular apolipoprotein onto lipoproteins, but most intracellular apolipoprotein remains non-lipoprotein bound. The constant presence of a large non-lipoprotein-bound pool suggests that apolipoprotein synthesis is not the rate limiting step in lipoprotein assembly or secretion.     

Novel Large Apolipoprotein E-Containing Lipoproteins of Density 1.006–1.060 g/ml in Human Cerebrospinal Fluid

Abstract: Although the critical role of apolipoprotein E (apoE) allelic variation in Alzheimer's disease and in the outcome of CNS injury is now recognized, the functions of apoE in the CNS remain obscure, particularly with regard to lipid metabolism. We used density gradient ultracentrifugation to identify apoE-containing lipoproteins in human CSF. CSF apoE lipoproteins, previously identified only in the 1.063–1.21 g/ml density range, were also demonstrated in the 1.006–1.060 g/ml density range. Plasma lipoproteins in this density range include low-density lipoprotein and high-density lipoprotein (HDL) subfraction 1 (HDL₁). The novel CSF apoE lipoproteins are designated HDL₁. No immunoreactive apolipoprotein A-I (apo A-I) or B could be identified in the CSF HDL₁ fractions. Large lipoproteins 18.3 ± 6.6 nm in diameter (mean ± SD) in the HDL₁ density range were demonstrated by electron microscopy. Following fast protein liquid chromatography of CSF at physiologic ionic strength, apoE was demonstrated in particles of average size greater than particles containing apoA-I. The largest lipoproteins separated by this technique contained apoE without apoA-I. Thus, the presence of large apoE-containing lipoproteins was confirmed without ultracentrifugation. Interconversion between the more abundant smaller apoE-HDL subfractions 2 and 3 and the novel larger apoE-HDL₁ is postulated to mediate a role in cholesterol redistribution in brain.     

Studies on the substrate specificity of human and pig lecithin: cholesterol acyltransferase: role of low-density lipoproteins

The substrate properties of low-density lipoprotein (LDL) fractions from human and pig plasma and of lipoprotein a [Lp(a)] upon incubation with either pig or human lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) were investigated and compared with those of pig high-density lipoproteins (HDL) or human HDL-3. The cholesterol esterification using purified native pig LDL-1, human LDL, or Lp(a) as a substrate was approximately 36-42% that of pig HDL or human HDL-3, while cholesteryl ester formation with pig LDL-2 was 41-47%. No significant difference was found in the substrate activity between pig HDL and human HDL-3, and between human LDL and Lp(a), respectively. After depletion of pig LDL-1, pig LDL-2, and human LDL from apolipoprotein A-I (apoA-I), cholesteryl ester formation decreased to about 22-28% of the value found with pig HDL. Depletion of human LDL from apolipoprotein E (apoE) did not result in significantly different esterification rates in comparison to native LDL. Total removal of non-apoB proteins from human LDL resulted in esterification rates of approximately 10-15% that of HDL. Readdition of apoA-I to all these LDL fractions produced solely in apoA-I-depleted LDL fractions an increase of cholesteryl ester formation, whereas in those LDL fractions that were additionally depleted from apoE and/or from apoC polypeptides, a further decrease in the esterification rate occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

IR spectroscopic study of phospholipid emulsions containing cholesteryl oleate

As models for the lipid organization of low density lipoproteins (LDL), protein-free aqueous emulsions are prepared from dimyristoyl phosphatidyl choline (DMPC), dipalmitoyl phosphatidyl choline (DPPC), and cholesteryl oleate (CO). Aqueous dispersions containing these lipids are sonicated and yield stable particles with diameters varying between 20 and 40 nm as measured through electron microscopy. IR spectroscopy shows that emulsions consisting of DMPC, DPPC, and CO at 3/1/1 and 1/1/1 ratios undergo specific thermal transitions, depending on their composition, that can be assigned to the phospholipids forming the surface layer of the emulsion particles and to core-located CO. However, at the 1/3/1 DMPC/DPPC/CO ratio this lipid system exhibits an order-disorder transition of the mixed phospholipids with no significant transition associated with core-located CO. Observation of the methylene C&bond;H and C&bond;D stretching modes of nondeuterated and deuterated lipids enables the packing characteristics and conformational order of each lipid to be monitored separately. The transition temperature changes compared to the temperatures for the analogous transitions in neat CO and CO-free phospholipid vesicles suggest the existence of interactions between CO and the above phospholipids in the ternary emulsion particles; these interactions are stronger at the 1/3/1 DMPC/DPPC/CO ratio. The results show that interactions between core and surface phases are dependent on the emulsion lipid composition and that these findings may be extended to native lipoproteins.     

Characterization of four lipoprotein classes in human cerebrospinal fluid.

Lipoprotein metabolism in brain has not yet been fully elucidated, although there are a few reports concerning lipids in the brain and lipoproteins and apolipoproteins in the cerebrospinal fluid (CSF). To establish normal levels of lipoproteins in human CSF, total cholesterol, phospholipids, and fatty acids as well as apolipoprotein E (apoE) and apoA-I levels were determined in CSF samples from 216 individuals. For particle characterization, lipoproteins from human CSF were isolated by affinity chromatography and analyzed for size, lipid and apolipoprotein composition. Two consecutive immunoaffinity columns with antibodies, first against apoE and subsequently against apoA-I, were used to define four distinct lipoprotein classes. The major lipoprotein fraction consisted of particles of 13;-20 nm containing apoE and apoA-I as well as apoA-IV, apoD, apoH, and apoJ. In the second particle class (13;-18 nm) mainly apoA-I and apoA-II but no apoE was detected. Third, there was a small number of large particles (18;-22 nm) containing no apoA-I but apoE associated with apoA-IV, apoD, and apoJ. In the unbound fraction we detected small particles (10;-12 nm) with low lipid content containing apoA-IV, apoD, apoH, and apoJ. In summary, we established lipid and apolipoprotein levels in CSF in a large group of individuals and described four distinct lipoprotein classes in human CSF, differing in their apolipoprotein pattern, lipid composition, and size. On the basis of our own data and previous findings from other groups, we propose a classification of CSF lipoproteins.     

Series of Concentration-Induced Phase Transitions in Cholesterol/Phosphatidylcholine Mixtures

In lipid membranes, temperature-induced transition from gel-to-fluid phase increases the lateral diffusion of the lipid molecules by three orders of magnitude. In cell membranes, a similar phase change may trigger the communication between the membrane components. Here concentration-induced phase transition properties of our recently developed statistical mechanical model of cholesterol/phospholipid mixtures are investigated. A slight (<1%) decrease in the model parameter values, controlling the lateral interaction energies, reveals the existence of a series of first- or second-order phase transitions. By weakening the lateral interactions first, the proportion of the ordered (i.e., superlattice) phase (A_reg) is slightly and continuously decreasing at every cholesterol mole fraction. Then sudden decreases in A_reg appear at the 0.18–0.26 range of cholesterol mole fractions. We point out that the sudden changes in A_reg represent first- or second-order concentration-induced phase transitions from fluid to superlattice and from superlattice to fluid phase. Sudden changes like these were detected in our previous experiments at 0.2, 0.222, and 0.25 sterol mole fractions in ergosterol/DMPC mixtures. By further decreasing the lateral interactions, the fluid phase will dominate throughout the 0.18–0.26 interval, whereas outside this interval sudden increases in A_reg may appear. Lipid composition-induced phase transitions as specified here should have far more important biological implications than temperature- or pressure-induced phase transitions. This is the case because temperature and pressure in cell membranes are largely invariant under physiological conditions.     

The lipidation by hepatocytes of human apolipoprotein A-I occurs by both ABCA1-dependent and -independent pathways

The pathways of hepatic intra- and peri-cellular lipidation of apolipoprotein A-I (apoA-I) were studied by infecting primary mouse hepatocytes from either apoA-I-deficient or ABCA1-deficient mice with a recombinant adenovirus expressing the human apoA-I (hapoA-I) cDNA (endo apoA-I) or incubating the hepatocytes with exogenously added hapoA-I (exo apoA-I) and examining the hapoA-I-containing lipoproteins formed. The cells, maintained in serum-free medium, were labeled with [(3)H]choline, and the cell medium was separated by fast protein liquid chromatography or immunoprecipitated to quantify labeled choline phospholipids specifically associated with hapoA-I. With the apoA-I-deficient hepatocytes, the high density lipoprotein fraction formed with endo apoA-I contained proportionally more phospholipids than that formed with exo apoA-I. However, the lipoprotein size and electrophoretic mobility and phospholipid profiles were similar for exo apoA-I and endo apoA-I. Taken together, these data demonstrate that a significant proportion of hapoA-I is secreted from hepatocytes in a phospholipidated state but that hapoA-I is also phospholipidated peri-cellularly. With primary hepatocytes from ABCA1-deficient mice, the expression and net secretion of adenoviral-generated endogenous apoA-I was unchanged compared with control mice, but (3)H-phospholipids associated with endo apoA-I and exo apoA-I decreased by 63 and 25%, respectively. The lipoprotein size and electrophoretic migration and their phospholipid profiles remained unchanged. In conclusion, we demonstrated that intracellular and peri-cellular lipidation of apoA-I represent distinct and additive pathways that may be regulated independently. Hepatocyte expression of ABCA1 is central to the lipidation of newly synthesized apoA-I but also contributes to the lipidation of exogenous apoA-I. However, a significant basal level of phospholipidation occurs in the absence of ABCA1.     

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.     

Role of lipids in the Neurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions.

The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and the cel-(Tw 40) mutant of Neurospora crassa. The cel-(Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at approximately 34 degrees C) had approximately twice the saturated fatty acid content of w/t organisms grown at approximately 22 degrees C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived from w/t and cel-(Tw 40) occur at -31 and -11 degrees C, respectively. The heats of transition (delta H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol in Neurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. Intact Neurospora, whether w/t or cal-(Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 degrees C was observed in intact cells and extracted phospholipids of both w/t and cel-(Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (delta H congruent to 1 cal/g), and was reversible.