Interaction of plasma apolipoproteins with lipid monolayers.

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidy[14C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipids by apolipoprotein C-III. The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface. Although apolipoprotein A-I and arginine-rich protein gave surface pressure increases, phospholipid was only slightly removed fromthe interface by the addition of liposomes. Based on these findings, we conclude that the apolipoproteins C interact specifically with phosphatidylcholine at the interface. This interaction is important as it relates to the transfer of the apolipoproteins C and phospholipids from very low density lipoproteins to other plasma lipoproteins. The addition of human plasma high density lipoproteins or very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of phosphatidyl[14C]choline from the interface 3--4 fold. Low density lipoproteins did not affect the rate of decrease. During lipolysis of very low density lipoproteins to the subphase increased the apolipoprotein C-mediated removal of with the lipid monolayer. Lipolysis experiments were performed in a monolayer trough containing a surface film of egg phosphatidyl[14C]choline and a subphase of very low density lipoproteins and bovine serum albumin. Lipolysis was initiated by the addition of purified milk lipoprotein lipase to the subphase. As a result of lipolysis, there was a decrease in surface radioactivity of phosphatidylcholine. The pre-addition of high density lipoproteins decreased the rate of decrease in surface radioactivity...     

Hepatic lipase hydrolysis of lipid monolayers. Regulation by apolipoproteins

A monolayer technique was used to study the substrate specificity of hepatic lipase (HL) and the effect of surface pressure and apolipoproteins on hydrolysis of lipid monolayers by this enzyme. HL hydrolyzed readily phosphatidylethanolamine monolayers. Pure trioctanoylglycerol was found to be a poor substrate but when progressively diluted with nonhydrolyzable 1,2-didodecanoylphosphatidylcholine hydrolysis of triacylglycerol by HL reached maximum at a molar ratio of 1:1 triacylglycerol to phosphatidylcholine. The activation of triacylglycerol hydrolysis was not due to altered penetration of HL. The surface pressure optimum of HL for the hydrolysis of phosphatidylethanolamine monolayers was broad between 12.5 and 25 mN/m. When apolipoprotein E was injected beneath the monolayer of phosphatidylethanolamine prior to enzyme addition, a 3-fold activation of HL was observed at surface pressures equal to or below 15 mN/m. Below surface pressures of 20 mN/m apolipoprotein E did not affect the penetration of HL into the lipid-water interface. Apolipoprotein E slightly activated the hydrolysis of triacylglycerol by HL at 10 mN/m. At a high surface pressure of 25 mN/m all apolipoproteins tested (apolipoproteins A-I, A-II, C-I, C-II, C-III, and E) inhibited the penetration into and HL activity on phosphatidylethanolamine At 18.5 mN/m all apolipoproteins except apolipoprotein E inhibited the hydrolysis of triacylglycerol in the triacylglycerol:phosphatidylcholine mixed film. Based on these results we present a hypothesis that phospholipid present in apolipoprotein E-rich high density lipoprotein-1 and triacylglycerol in intermediate density lipoprotein would be preferred substrates for HL.     

The interaction of lipolysis products of very low density lipoprotein with plasma high density lipoprotein (HDL): perfusate HDL with plasma HDL subfractions

The nature of the interaction of high density lipoproteins (HDL), formed during lipolysis of human very low density lipoprotein (VLDL) by perfused rat heart, with subfractions of human plasma HDL was investigated. Perfusate HDL, containing apoliproproteins (apo) E, C-II, and C-III but no apo A-I or A-II, was incubated with a subfraction of HDL (HDL-A) containing apo A-I and A-II, but devoid of apo C-II, C-III, and E. The products of the incubation were resolved by heparin-Sepharose or hydroxylapatite chromatography under conditions which allowed the resolution of the initial HDL-A and perfusate HDL. The fractions were analyzed for apolipoprotein content and lipid composition and assessed for particle size by electron microscopy. Following the incubation, the apo-E-containing lipoproteins were distinct from perfusate HDL since they contained apo A-I as a major component and apo C-II and C-III in reduced proportions. However, the HDL-A fraction contained apo C-II and C-III as major constituents. Associated with these changes in apolipoprotein composition, the apo-E-rich lipoproteins acquired cholesteryl ester from the HDL-A fraction and lost phospholipid to the HDL-A fraction. The HDL-A fraction maintained a low unesterified cholesterol/phospholipid molar ratio (0.23), while the apo-E-containing lipoproteins possessed a high ratio (0.75) characteristic of the perfusate HDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.     

Human chylomicron apolipoprotein metabolism.

Chylomicron apolipoprotein metabolism was studied utilizing chylomicrons isolated from the pleural fluid of a patient with a recurrent chylous pleural effusion. Chylomicrons contained apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, E, and albumin. Following intravenous injection of [¹²⁵I] chylomicrons, almost all of the A apolipoprotein radioactivity was recovered in high density lipoproteins, while only a small amount of the B apolipoprotein radioactivity was recovered in low density lipoproteins. These observations indicate that intestinal chylomicron A apolipoproteins serve as precursors for plasma high density lipoprotein A apolipoproteins and only a small fraction of chylomicron apolipoprotein B is metabolized to form low density lipoprotein apolipoprotein B.     

Activation of hepatic lipase catalyzed phosphatidylcholine hydrolysis by apolipoprotein E

The effect of apolipoproteins A-I, A-II, C-II, C-III and E on the hydrolysis of phosphatidylcholine and triacylglycerol by hepatic lipase was studied. Hepatic lipase catalyzed phospholipid hydrolysis was 1.8-fold activated by apolipoprotein E while the other apolipoproteins did not affect the hydrolysis by this enzyme. Triacylglycerol hydrolysis by hepatic lipase was 1.5-fold activated by apolipoprotein E while the other apolipoproteins inhibited hepatic lipase. These results suggest that lipoproteins containing apolipoprotein E may be preferred substrates for hepatic lipase.     

Adsorption of apolipoprotein A-IV to phospholipid monolayers spread at the air/water interface. A model for its labile binding to high density lipoproteins.

The mechanisms that mediate the labile binding of apolipoprotein A-IV (apoA-IV) to high density lipoproteins (HDL) are not known. We therefore used a surface balance and surface radioactivity detector to investigate the adsorption of apoA-IV to egg phosphatidylcholine monolayers spread at the air/water interface. ApoA-IV bound rapidly and reversibly to phospholipid monolayers and generated a maximum increase in surface pressure of 19 millinewtons (mN)/m at a subphase concentration of 2 x 10(-5) g/dl. Binding decreased linearly with increasing initial surface pressure; at pressures greater than 28-29 mN/m, apoA-IV could no longer penetrate the lipid monolayer. The area occupied by the amino acid residues in apoA-IV reached an unusually low limiting molecular area of 10-12 A2/residue at surface saturation. The surface pressure of native HDL3 was calculated to be 33 mN/m, and it rapidly decreased with the action of lecithin:cholesterol acyltransferase on the particle surface. We conclude that the surface activity of apoA-IV is lower than that of any other human apolipoprotein; its binding and surface conformation are particularly sensitive to pressure; and at saturation, a significant portion of the molecule is excluded from the interface. The exclusion pressure of apoA-IV may be only slightly lower than the surface pressure of HDL; in vivo, the action of lecithin:cholesterol acyltransferase and lipid transfer proteins may cause the HDL3 surface pressure to oscillate about a narrow range that spans the exclusion pressure of apoA-IV. The resultant labile association of apoA-IV and HDL may be of central importance to its role in lipoprotein metabolism.     

Distribution of C apolipoproteins between the vascular and lymph compartments of the rat

This study has investigated the kinetics of transfer of C apolipoproteins between the vascular and lymph compartments of the rat. Very-low-density lipoprotein, labeled with [125I]apolipoprotein C, was injected intravenously into lymph duct-cannulated rats and the redistribution of radioactivity between lymph and plasma followed at frequent intervals for 3 h. Equilibration between the two compartments was rapid (10-15 min), and thereafter removal from both compartments continued at similar rates. Specific radioactivity determinations showed that lymph C-III-0, C-III-3, and C-III-2,1 apolipoproteins rapidly reached values identical to those of corresponding plasma C apolipoproteins and the interrelationship between the curves were consistent with precursor-product relationships in which all, or most, of the product (lymph apolipoprotein C-III) was derived from the precursor (plasma). However, the specific radioactivity curves for C-II peptide did not cross; the lower value for lymph C-II apolipoprotein suggests that, unlike C-III apolipoproteins, a substantial proportion (approx. 40%) of lymph C-II peptide is not derived from the plasma compartment. The most likely source of the unlabeled lymph apolipoprotein C-II is synthesis and secretion from the intestine.     

A comparative study on the removal of cellular lipids from Landschütz ascites cells by human plasma apolipoproteins.

The effects of human plasma lipoprotein-proteins on the removal of cellular lipids from Landschütz ascites cells were studied. Cellular lipids were labeled by injecting mice previously injected with ascites with either [3H]cholesterol or [3H]choline. Apoproteins from very low density (apoC-I, C-II, and C-111) and high density (apoA-I and A-II) lipoproteins were used. Each of the apoproteins alone was ineffective in removing cellular [3H]cholesterol. However, when synthetic phosphatidylcholines of known composition were added to each apoprotein and the experiments were repeated using either apoprotein-lipid mixtures or ultracentrifugally isolated complexes, the removal of sterol was considerably enhanced. Complexes of saturated phosphatidylcholines with apoA-II, apoC-I, or apoC-III were the most effective in releasing cellular sterol. Apoprotein-phospholipid complexes were much less effective in removing cellular [3H]phosphatidylcholine than the free apoproteins; apoA-I and apoC-I were the best of the five apoproteins studied. When a comparison was made of the adsorption of iodinated apoproteins to ascites cells, 3 to 4 times more apoA-II and apoC-III were bound than apoA-I. The binding of apoproteins was time and temperature dependent. Approximately 50% of the radioactivity that remained in the washed cells was removed with trypsin. To determine if the counts remaining in the trypsin-treated cells were internalized, identical experiments were performed using human erythrocytes, cells that do not exhibit pinocytosis. Again, approximately 50% of the radioactivity of the iodinated apoproteins was not released by trypsin. Succinylation of apoA-II not only destroys its phospholipid-binding properties but also its adsorption to red cells. These results suggest that the plasma apoproteins differ in their ability to remove cellular lipids and bind to both ascites and red cell membranes, and possibly to specific phospholipids, in such a way that only a part of the apoprotein is degraded with proteases.     

Apolipoproteins of high, low, and very low density lipoproteins in human bile

We tested the hypothesis that apolipoproteins, the protein constituents of plasma lipoproteins, are secreted into bile. We examined human gallbladder bile obtained at surgery (N = 54) from subjects with (N = 44) and without (N = 10) gallstones and hepatic bile collected by T-tube drainage (N = 9) after cholecystectomy. Using specific radioimmunoassays for human apolipoproteins A-I and A-II, the major apoproteins of high density lipoproteins, for apolipoproteins C-II and C-III, major apoproteins of very low density lipoproteins, and for apolipoprotein B, the major apoprotein of low density lipoproteins, we found immunoreactivity for these five apolipoproteins in every bile sample studied in concentrations up to 10% of their plasma values. Using double immunodiffusion, we observed complete lines of identity between bile samples and purified apolipoproteins A-I, A-II, or C-II. Using molecular sieve chromatography, we found identical elution profiles for biliary apolipoproteins A-I, A-II and B and these same apolipoproteins purified from human plasma. When we added high density lipoproteins purified from human plasma to lipoprotein-free solutions perfusing isolated rat livers, we detected apolipoproteins A-I and A-II in bile. Similarly, when we added low density lipoproteins purified from human plasma to lipoprotein-free solutions perfusing isolated livers of rats treated with ethinyl estradiol in order to enhance hepatic uptake of low-density lipoproteins, we found apolipoprotein B in bile. These data indicate that apolipoproteins can be transported across the hepatocyte and secreted into bile.     

Specificity of the lipid-binding domain of apoC-II for the substrates and products of lipolysis

Functional similarities between colipase and apolipoprotein C-II (apoC-II) in activating lipases suggest that apoC-II may, like colipase, preferentially interact with interfaces containing the substrates and products of lipolysis. To test this hypothesis, the binding of a peptide comprising residues of the cofactor implicated in lipid binding, apolipoprotein C-II(13-56), and, to a lesser extent, apoC-II, to monomolecular lipid films was characterized. The lipids used were a diacylphosphatidylcholine, a diacylglycerol, and a fatty acid. The peptide had an affinity for the argon-buffer interface and for all lipids consistent with a dissociation constant of <10 nM. Changes in surface pressure accompanying peptide binding were comparable to those reported for native apoC-II and indicate peptide miscibility with each of the lipids tested. The capacity of the surfaces to accommodate the peptide decreased with increasing lipid concentration in the interface, indicating competition between lipid and peptide for interfacial occupancy. At a lipid acyl chain density of 470 pmol/cm2, or 35 A2 per acyl chain, a lower limit of peptide adsorption was reached with all lipids. The limiting level of adsorption to phosphatidylcholine was only 1 pmol/cm2 compared with 6;-7 pmol/cm2 for fatty acid and diacylglycerol. Similar results were obtained with apoC-II.The difference in the extent of protein adsorption to lipid classes suggests that the distribution of apoC-II among lipoproteins will depend on their lipid composition and surface pressure.     

Retention of apolipoprotein B and cholesterol by perfused heart during lipolysis of very-low-density lipoprotein

The fate and mechanism of removal of apolipoproteins and lipids of human very-low-density lipoproteins were determined in the perfused rat heart. Approx. 50% of the VLDL triacylglycerol was hydrolyzed during a 2 h perfusion. Phospholipid phosphorus, apolipoproteins C-II, C-III and E were quantitatively recovered in the medium. However, there was a loss of unesterified (17 +/- 6%) and esterified (19 +/- 8%) cholesterol from the perfusion medium. Apolipoprotein B was retained by the heart, as determined by the loss of immunoassayable apolipoprotein B (30 +/- 5%) or the uptake of 125I-labelled apolipoprotein of VLDL (9 +/- 2%) from the perfusion medium. The discrepancy in the two methods for estimating apolipoprotein removal was shown to be due to the modification of apolipoprotein B-containing lipoproteins, which was such that they were no longer precipitated with antibodies to apolipoprotein B. The labelled apolipoprotein B, retained by the heart, could be partially released by perfusion of the heart with buffer containing heparin (14 +/- 2%) or trypsin (50 +/- 2%). Labelled apolipoprotein uptake by the heart was reduced by 90% when lipoprotein lipase was first released by heparin or when VLDL was treated with 1,2-cyclohexanedione to modify arginine residues of apolipoproteins. Very little extensive degradation of the apoprotein to low molecular weight material occurred during the 2 h perfusion, since 95% of the tissue label was precipitated by trichloroacetic acid. It is concluded that there is retention of apolipoprotein B, cholesteryl ester and cholesterol by the perfused heart during catabolism of VLDL. The data are consistent with the concept that the retention of apolipoprotein B requires membrane-bound lipoprotein lipase or an interaction with the cell surfaces that is modified by heparin. The overall process also involves arginine residues of apolipoproteins. At least 50% of the labelled apolipoprotein retained in the tissue is associated with lipoprotein lipase and other cell surface sites, while the remainder may be taken up by the cells.     

Effect of lipid particle size on association of apolipoproteins with lipid

Triolein particles stabilized with egg yolk phosphatidylcholine monolayer were prepared with two different diameters: 26.7 +/- 3.9 and 229 +/- 80 nm. All the phosphatidylcholine molecules in those particles were readily digested by phospholipase A2 while only the molecules in the outer leaflet of phosphatidylcholine unilamellar vesicles were hydrolyzed under the same conditions. Binding of human plasma apolipoproteins A-I, A-II, C-II, and C-III2 to the particles was studied by two independent techniques: (i) rapid gel permeation chromatography and (ii) ultracentrifugation. All four apolipoproteins bound to the small and large particles in a saturable manner without altering their gross structure, and were displaced by equivalent molecules. The dissociation constant of apolipoprotein A-I for the large particle was 3.17 X 10(-6) M and 4.24 X 10(-6) M by methods (i) and (ii), respectively. These values were more than 10-fold greater than those for the small particles (2.0 X 10(-7) and 1.6 X 10(-7) M, respectively). In contrast, apolipoproteins A-II, C-II, and C-III2 bound to the large particles as strongly as to the small particles with dissociation constants of 2.4-6.8 X 10(-7), 4.5-10.7 X 10(-7), and 5.3-10.7 X 10(-7) M, respectively. The maximum binding level was of a similar order for each of the four apolipoproteins with both lipid particles when they were compared on the basis of amino acids per phospholipid. These results suggest that the apolipoproteins share common binding sites on the lipid particles, and are consistent with the characteristic distribution of apolipoproteins A-I and C among various classes of lipoproteins in plasma.     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and β-casein with phospholipids

The conformations adopted by β-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being α-helical and the β-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the β-casein is in a compressed state whereas the apoprotein is extended as α-helices in the plane of the interface. The chain-length dependences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

A comparison of the interfacial interactions of the apoprotein from high density lipoprotein and beta-casein with phospholipids.

The conformations adopted by beta-casein and the total apoprotein from serum high density lipoprotein when spread at the air-water interface are compared; the monolayer data are consistent with the apoprotein being alpha-helical and the beta-casein being disordered with segments distributed in loops and trains. The penetration of these hydrophobic proteins into phosphatidylcholine monolayers in different physical states was investigated. More protein can penetrate into monolayers when they are in the liquid-expanded state; for penetration at constant total surface area the lateral compressibility of the lipid is an important factor. The charge and conformation of the polar group of the phospholipid does not have a major influence on the interaction. The mixed films of lipid and protein have a mosaic structure; probably the beta-casein is in a compressed state whereas the apoprotein is extended as alpha-helices in the plane of the interface. The chain-length depedences of the interaction of the apoprotein with phosphatidylcholine monolayers and bilayers are different; when the apoprotein binds to bilayers of shorter-chain phosphatidylcholines it alters the shape of the lipid-water interface whereas with monolayers the interface remains planar throughout.     

The immunochemical detection of apoprotein dissociation from particles of very low-density lipoproteins in human blood plasma]

The dissociation of very-low-density lipoprotein (VLDL) apoproteins was studied using immunochemical approaches. The analysis of monospecific antibody binding to apo E, C-II and C-III on VLDL surface showed low apoprotein accessibility for the antibodies while the accessibility of apo C-II and C-III in solution was complete. Lipoprotein preparation dilution resulted in increasing of apo E and C-II accessibility. It was suggested that apoprotein dissociation led to apoprotein cluster dissolving on VLDL surface and higher antigen determinant accessibility. The findings confirmed previous theoretical analysis of apoprotein dissociation.     

Activation of lipoprotein lipase by N-alpha-palmitoyl (56-79) fragment of apolipoprotein C-II

The effect of apolipoprotein C-II (apoC-II) and a synthetic fragment of apoC-II corresponding to residues 56-79 on the lipoprotein lipase (LpL) catalyzed hydrolysis of trioleoylglycerol in a monolayer of egg phosphatidylcholine and of dipalmitoylphosphatidylcholine vesicles was examined. Synthetic peptide 56-79, which does not associate with lipid, did not activate LpL at surface pressures greater than 30 mN/m; apoC-II is active up to 34 mN/m. However, acylation of the NH2-terminus of peptide 56-79 with palmitoyl chloride gave nearly identical LpL activating properties as compared to apoC-II. We conclude that at high surface pressures the lipid-binding region of apoC-II (residues 44-55) plays an essential role in LpL activation.     

Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry

The interactions between phospholipid molecules in suspensions have been studied by using mass spectrometry. Electrospray mass spectra of homogeneous preparations formed from three different phospholipid molecules demonstrate that under certain conditions interactions between 90 and 100 lipid molecules can be preserved. In the presence of apolipoprotein C-II, a phospholipid binding protein, a series of lipid molecules and the protein were observed in complexes. The specificity of binding was demonstrated by proteolysis; the resulting mass spectra reveal lipid-bound peptides that encompass the proposed lipid-binding domain. The mass spectra of heterogeneous suspensions and their complexes with apolipoprotein C-II demonstrate that the protein binds simultaneously to two different phospholipids. Moreover, when apolipoprotein C-II is added to lipid suspensions formed with local concentrations of the same lipid molecule, the protein is capable of remodeling the distribution to form one that is closer to a statistical arrangement. These observations demonstrate a capacity for apolipoprotein C-II to change the topology of the phospholipid surface. More generally, these results highlight the fact that mass spectrometry can be used to probe lipid interactions in both homogeneous and heterogeneous suspensions and demonstrate reorganization of the distribution of lipids upon surface binding of apolipoprotein C-II.     

Enhancement of non-polar lipid transfer reaction through stabilization of substrate lipid particles with apolipoproteins

Transfer of lipids was studied between human plasma low density lipoproteins (LDL) and triolein particles coated with an egg phosphatidylcholine monolayer, with diameter of 27 +/- 4 nm. The lipid particles were unstable and seemed to aggregate to LDL when incubated with LDL either in the presence or the absence of bovine serum albumin. Human apolipoproteins A-I, A-II, C-II, C-III, and E stabilized the lipid particles and completely prevented this process. Cholesterol rapidly appeared in the lipid particles to reach homogeneous distribution among the phospholipid surfaces of LDL and the lipid particles regardless of whether apolipoproteins were present or absent. Cholesteryl ester spontaneously appeared in the lipid particles to some extent in the absence of the apolipoproteins, and human plasma lipid transfer protein enhanced this reaction only to a very limited extend. When the lipid particles were stabilized with the apolipoproteins, spontaneous cholesteryl ester transfer was minimized and the lipid transfer protein catalyzed the transfer of cholesteryl ester markedly. There was no specific difference among the apolipoproteins in stabilizing the particles and enhancing the transfer reaction. Reciprocal decrease in volume of triglyceride was observed at the same time in the lipid particles until the relative content of cholesteryl ester in the cores of LDL was the same as in the lipid particles. The kinetics of the cholesteryl ester and triglyceride transfer was consistent with the model that the reaction is bidirectional in equilibrium and takes both non-polar lipids as substrate in a single pool.     

The major low molecular weight apolipoprotein from normal and hyperlipidemia atherosclerosis-prone (LAP) Japanese quail

The low molecular weight (LMW) apolipoprotein of apo C plays an important role in the metabolism of triglyceride-rich lipoproteins. This study aimed at a characterization of the major LMW apolipoproteins from normal quail strain, and also from LAP (hyperlipidemia atherosclerosis-prone) strain to identify its genetic disorder. The major LMW apoprotein cDNA clone from normal quail comprised of approximately 500 bp, and encoded polypeptide of 78 amino acid residues containing 57 amino acids as a mature apolipoprotein. Although the quail LMW apoprotein showed a low homology to either apo C-I, C-II, or C-III of other animals, it retained a well-developed amphipathic alpha-helix structure. There was no difference in the deduced primary structure of the quail LMW apoprotein between LAP and normal strain. An analysis of the mRNA expression showed that the quail LMW apoprotein was only expressed in the liver of both LAP and normal Japanese quail. No difference was noted in the hepatic expression of the quail LMW apoprotein mRNA between normal and LAP strains with neither normal nor atherogenic dietary conditions. The structure and expression of the major LMW apoprotein thus had no relevance to higher susceptibility of LAP strain to the experimental atherosclerosis.