Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.

Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 to 3800 microseconds were observed, suggesting that band 3 exists in different aggregate states within the plane of the membrane. The oxidizing agent phenylhydrazine was used to induce hemichrome formation within intact erythrocytes. The immobilization of band 3 in membranes prepared from these erythrocytes suggests that the binding of hemichromes induces clustering of band 3. The addition of purified hemichromes to erythrocyte ghosts leads to a similar effect. We have also examined the mobility of the cytoplasmic domain of band 3. This region was labeled indirectly using a phosphorescently labeled antibody which binds to an epitope within the cytoplasmic domain. We observed very rapid motion of the cytoplasmic region of band 3, which was only partially restricted upon hemichrome binding. This suggests that the integral and cytoplasmic domains of band 3 may be independently mobile.     

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.     

The effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors.     

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.     

Apolipoprotein specificity of the chicken oocyte receptor for low and very low density lipoproteins: lack of recognition of apolipoprotein VLDL-II

At onset of egg-laying in the chicken, plasma levels of apolipoprotein VLDL-II (apoII) increase dramatically, suggesting a function of apoII in yolk deposition of triglyceride-rich lipoproteins. Thus, the possibility that this female-specific homodimeric protein (Mr of subunit, 9500) is recognized by the oocyte receptor for low and very low density lipoproteins was investigated. ApoII was purified from very low density lipoproteins by a novel, rapid procedure and reconstituted with egg phosphatidylcholine (PC) by detergent-dialysis. The resulting discoidal apoII/PC lipoprotein particles contained 3 mg of apoII per mg of PC and had a buoyant density of 1.062 g/ml. The ability of apoII/PC, as well as of physiological particles containing apoII but devoid of apolipoprotein B (apoB), namely high density lipoproteins (HDL) from laying hens, to interact with the oocyte receptor was tested. Both of these ligands failed to show saturable high affinity binding, in contrast to the apoB-containing ligands, low and very low density lipoproteins. Furthermore, neither laying-hen HDL which contain apoII and apoA-I nor apoII/PC were able to displace receptor-bound apoB-containing lipoproteins, as shown in competitive binding assays as well as by ligand blotting. Thus, we conclude that apoB, but not apoII, participates in binding and uptake of very low density lipoproteins via receptor-mediated endocytosis by growing chicken oocytes.     

Apolipoproteins, membrane cholesterol domains, and the regulation of cholesterol efflux.

Published data related to both cell membrane biology and apolipoprotein structure are reviewed and used to formulate a new model describing the mechanisms of cholesterol efflux from cell plasma membrane to high density lipoprotein (HDL) particles. The central premise of this model is the existence of heterogenous domains of cholesterol within plasma membranes. We propose that cholesterol efflux from cell membranes is influenced by three factors: 1) the distribution of cholesterol between cholesterol-rich and cholesterol-poor membrane domains, 2) the diffusion of cholesterol molecules through the extracellular unstirred water layer, and 3) the transient interaction of segments of the amphipathic helix of the HDL apolipoprotein with cholesterol-poor membrane domains resulting in enhanced cholesterol efflux.     

Independent flexible motion of submolecular domains of the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum measured by time-resolved fluorescence depolarization of site-specifically attached probes

The Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was site-specifically labeled with either N-(1-anilinonaphth-4-yl)maleimide (ANM) or 5-[[(iodoacetamido)-ethyl]amino]naphthalene-1-sulfonate (IAEDANS), and the segmental motion of submolecular domains of the ATPase molecule was examined by means of time-resolved and steady-state fluorescence anisotropy measurements. The ANM-binding domain showed wobbling with a rotational relaxation time phi = 69 ns in the absence of free Ca2+ without any independent wobbling of the ANM moiety. The IAEDANS-binding domain showed a significantly slower wobbling with phi = 190 ns in the absence of Ca2+. The present results demonstrated for the first time that the ATPase molecule is composed of distinct domains whose mobilities are considerably different from each other. The binding of Ca2+ to the transport site increased the segmental motion of ANM-labeled domain, leading to a phi value of 65 ns. Solubilization of the ANM-labeled SR membranes by deoxycholate led to a further increase in the segmental flexibility (phi = 48 ns in the absence of free Ca2+), indicating that the mobility of the ANM-binding domain was considerably restricted through interaction with the membrane. The mobility of the ANM-binding domain of solubilized ATPase was also increased to some extent upon binding of Ca2+.     

Binding of apolipoprotein A-I and A-II after recombination with phospholipid vesicles to the high density lipoprotein receptor of luteinized rat ovary

To determine the apolipoprotein specificity of high density lipoprotein (HDL) receptor, apolipoprotein A-I (apo-AI) and apolipoprotein A-II (apo-AII) purified from high density lipoprotein3 (HDL3) were reconstituted into dimyristoyl phosphatidylcholine vesicles (DMPC) and their ability to bind to luteinized rat ovarian membranes was examined. Both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC were shown to bind to ovarian membranes with Kd = 2.87 and 5.70 micrograms of protein/ml, respectively. The binding of both 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC was inhibited by unlabeled HDL3, apo-A-I.DMPC, apo-A-II.DMPC, apo-C-I.DMPC, apo-C-II.DMPC, apo-C-III1.DMPC, and apo-C-III2.DMPC, but not by DMPC vesicles, bovine serum albumin.DMPC or low density lipoprotein. Since the binding labeled apo-A-I.DMPC and apo-A-II.DMPC was inhibited by the DMPC complexes of apo-C groups, the direct binding of 125I-apo-C-III1.DMPC was also demonstrated with Kd = 9.6 micrograms of protein/ml. In addition, unlabeled apo-A-I.DMPC, and apo-A-II.DMPC, as well as apo-C.DMPC, inhibited 125I-HDL3 binding. 125I-apo-A-I, 125I-apo-A-II, and 125I-apo-C-III1 in the absence of DMPC also bind to the membranes. These results suggest that HDL receptor recognizes apolipoprotein AI, AII, and the C group and that the binding specificity of the reconstituted lipoproteins is conferred by their apolipoprotein moiety rather than the lipid environment. In vivo pretreatment of rats with human chorionic gonadotropin resulted in an increase of 125I-apo-A-I.DMPC, 125I-apo-A-II.DMPC, and 125I-apo-C-III1.DMPC binding activities. However, no induction of binding activity was observed when the apolipoprotein was not included in DMPC vesicles. An examination of the equilibrium dissociation constant and binding capacity for 125I-apo-A-I.DMPC and 125I-apo-A-II.DMPC after human chorionic gonadotropin treatment revealed that the increase in binding activity was due to an increase in the number of binding sites rather than a change in the binding affinity. These results further support our contention that apo-A-I, apo-A-II, and the apo-C group bind to HDL receptor. In conclusion, the HDL receptor of luteinized rat ovary recognizes apolipoproteins A-I, A-II, and the C group but not low density lipoprotein, and the binding is induced by human chorionic gonadotropin in vivo.     

Lipoprotein metabolism in the ovariectomized rat

The hyperlipoproteinemia observed after ovariectomy in rats was previously shown to be associated with increased concentrations of cholesterol, triglycerides, and apolipoproteins B, E, and C. In the present study, it was shown that increases in low density lipoproteins and high density lipoproteins were almost entirely responsible for the changes in plasma lipids and apolipoproteins after ovariectomy. The size of the low density lipoproteins and high density lipoproteins isolated from the plasma of ovariectomized rats as determined by agarose chromatography appeared to be somewhat different from that of control rats. Specifically, the apolipoprotein B appeared to be associated with somewhat smaller particles, whereas the apolipoprotein E from those rats appeared to be associated with larger particles than that of control rats. To determine the mechanism for the increased plasma low density lipoproteins, apolipoprotein B pool sizes and turnover rates were calculated and compared. In addition to an increased mass of low density lipoproteins in ovariectomized rats, the turnover rate of low density lipoproteins was increased almost twofold, indicating an increased low density lipoprotein synthesis and catabolism in those animals. We postulate that the increased low density lipoprotein levels of ovariectomized rats are due to an initial increased production of low density lipoproteins, followed by an enhanced catabolism of low density lipoproteins to establish a steady state at higher plasma low density lipoprotein concentrations.     

Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells

Cholesterol efflux was studied in cultured mouse adipose cells after preloading with low density lipoprotein cholesterol. Exposure to complexes containing human apolipoprotein A-IV and L-alpha-dimyristoylphosphatidylcholine (DMPC) as well as to human lipoprotein particles containing apolipoprotein A-IV but not apolipoprotein A-I and particles containing apolipoproteins A-IV and A-I showed that both artificial and native apolipoprotein A-IV-containing particles were able to promote cholesterol efflux at 37 degrees C as a function of time and concentration. The half-maximal concentration was found to be 0.3 X 10(-6) M for apolipoprotein A-IV.DMPC complexes. Binding experiments performed in intact cells at 4 degrees C with labeled apolipoprotein A-IV.DMPC complexes showed the existence of specific binding sites, with a Kd value of 0.32 x 10(-6) M and a maximal binding capacity of 223,000 sites/cell. By cross-competition experiments with labeled and unlabeled complexes containing apolipoprotein A-IV, A-I, or A-II, it appeared that all three apolipoproteins bind to the same cell-surface recognition sites. It is suggested that apolipoprotein A-IV, which is present in the interstitial fluid surrounding adipose cells in vivo at concentrations similar to those required in vitro for the promotion of cholesterol efflux, plays a critical role in cholesterol removal from peripheral cells.     

Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol

The distribution of apolipoprotein A-I, apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein in fasting normal human plasma was determined by two-dimensional electrophoresis followed by immunoblotting. The synthesis and transfer of labeled cholesteryl esters generated in plasma briefly incubated with [3H]cholesterol-labeled fibroblasts was followed in terms of the lipoprotein species containing these antigens. Following the early appearance of labeled free cholesterol in two pre beta-migrating apolipoprotein A-I species (Castro, G. R., and Fielding, C. J. (1988) Biochemistry 27, 25-29), labeled esters were first detected, after a 2-min delay, in a third pre beta-migrating species which also contained apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein. Pulse-chase experiments determined that label generated in this fraction was the precursor of at least a major part of labeled cholesteryl esters in the bulk of alpha-migrating high density lipoprotein. Over the maximum time course of these experiments (15 min, 37 degrees C), less than 10% of labeled cholesteryl esters were recovered in low or very low density lipoproteins separated by electrophoresis, immunoaffinity, or heparin-agarose chromatography. These data suggest channeling of cell-derived cholesterol and cholesteryl esters derived from it through a preferred pathway involving several minor pre beta-migrating lipoproteins to alpha-migrating high density lipoprotein.     

Interactions between human and carp (Cyprimus carpio) low density lipoproteins (LDL) and LDL receptors

1. We have compared the concentration and chemical composition of carp and human plasma lipoproteins and studied their interaction with human fibroblast LDL receptors. 2. The main lipoproteins in carp are of high density (HDL) in contrast to low density lipoproteins (LDL) in human. 3. Carp lipoproteins are devoid of apolipoprotein (apo) E, a major ligand for interaction with LDL receptors in mammals. 4. Carp very low density lipoproteins (VLDL) and LDL but not HDL nor apoA-I cross react with human LDL in their interaction with LDL receptors on human cultured fibroblasts. 5. Carp liver membranes possess high affinity receptors that are saturable and have calcium dependent ligand specificity (apoB and apoE) similar to human LDL receptor. Carp VLDL and LDL but not HDL nor its major apolipoprotein complexed to L-alpha-phosphatidylcholine dimyristoyl (apoA-I-DMPC) competed with the specific binding of human LDL to this receptor.     

The binding of low density lipoprotein by liver membranes in the pig.

Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of ¹²⁵I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL.     

Binding of intermediate density lipoproteins rich or poor in high molecular weight apolipoprotein B to rat liver membranes

Very low density lipoproteins rich or poor in high molecular weight apolipoprotein B (Bh-rich or Bh-poor VLDL, respectively) were prepared from rats fasted for 2 days and animals fasted and then refed for 2 days, respectively. Bh-rich or Bh-poor VLDL remnants (IDL) were also prepared by in vitro lipolysis of the corresponding VLDL preparations, and their apolipoprotein (apo) profile and lipid composition determined. Bh-rich IDL are richer in esterified cholesterol than Bh-poor IDL, but poorer in apoC and triglycerides. The binding of 125I-labeled Bh-rich IDL and 125I-labeled Bh-poor IDL to rat liver membranes was assessed by saturation-curve studies. Both types of IDL bound to high- and low-affinity sites on rat liver membranes. There were no significant differences between the binding of IDL produced from Bh-rich or Bh-poor VLDL to either the high- or low-affinity sites. However, by masking the low-affinity binding sites with saturating amounts of human high density lipoproteins 3 (HDL3), we were able to demonstrate that Bh-rich IDL bound to high-affinity binding sites with five times less affinity than Bh-poor IDL. These results show that saturating the low-affinity binding sites of rat liver membranes reveals differences in the binding abilities of lipoproteins to the high-affinity sites. Also, an analysis of apo and lipid compositions of the two types of IDL reveals that the apoBh contribution is likely to be responsible for differences in affinities of IDL for the high-affinity binding sites of rat liver membranes.     

Supermolecular organization of apolipoprotein E in solution]

The aggregation behaviour and stability in solution of apolipoprotein E (apoE) isolated from human blood plasma very low density lipoproteins were investigated. The equilibrium denaturation of fluorescein-labeled apoE by guanidine-hydrochloride determined by anisotropy and overall intensity of fluorescence, shift of the emission spectrum maximum and gel-chromatographic behaviour was characterized by reversibility, biphasity, apoE concentration dependence and the existence of native structure of the apoE monomer. The contribution of the long-living component to the kinetic dependence of fluorescence anisotropy in the presence of the 6 M denaturant increased with an increase in apoE concentration. The data obtained fit into the following scheme: oligomer (upon aging of the preparation) in equilibrium tetramer in equilibrium native monomer in equilibrium denaturated monomer. The presence in the tetrameric structure of apoE of two domains is postulated; one of those is formed by lipid-binding fragments during aggregation of individual molecules of apoE. Monoclonal antibody 3D12F11 (subclass IgG1) showed a high affinity for the apoE (Kd = 3.5 +/- 0.5 nM) without any effect on the apoprotein binding to heparin-Sepharose and apoE-induced destruction of dipalmitoylphosphatidylcholine liposomes. It is concluded that the 3D12F11 epitope is localized outside heparin- and lipid-binding sites of the apoprotein molecule.     

Abnormal apolipoprotein composition in alcoholic hepatitis

Alcoholic hepatitis leads to major derangements in lipoprotein metabolism. This study defines the characteristics of the abnormal high density lipoprotein and very low density lipoprotein in relation to the severity of the disease. In severely affected subjects very low density lipoprotein apolipoproteins were deficient in apolipoprotein E and apolipoprotein C. The concentration of high density lipoprotein was markedly reduced, although the proportion of high density lipoprotein 1 was substantially elevated when compared to normal subjects. High density lipoproteins were deficient in apolipoprotein AI and apolipoprotein AII but enriched in apolipoprotein E, apolipoprotein E complexes and apolipoprotein C, and contained a mixture of particles. The high density lipoprotein of subjects with alcoholic hepatitis contained a high proportion of material which bound to heparin affinity columns. This bound fraction contained a group of particles rich in apolipoprotein E, apolipoprotein E complexes and apolipoprotein C and was deficient in apolipoprotein AI and apolipoprotein AII. Examination by electron microscopy showed the presence of both discoidal and spherical particles, which varied in concentration according to the severity of the disease. Another fraction of high density lipoprotein, not bound to heparin, contained reduced amounts of apolipoprotein AI and apolipoprotein AII, consisted of disc-shaped particles and showed a higher esterified: free cholesterol ratio than the other high density lipoprotein fraction.     

Evidence for high density lipoproteins as the major apolipoprotein A-IV-containing fraction in normal human serum

The distribution of human apolipoprotein A-IV was studied in sera from normolipidemic fasting subjects by high performance gel filtration on a Superose 12 HR column. The major part of apolipoprotein A-IV eluted in the range of the apolipoprotein A-I peak, and distributed mainly in the large-size high density lipoprotein subfractions. Only a small peak or a shoulder on the main fraction appeared in the elution volume of free apolipoprotein A-IV. To investigate the relation of apolipoprotein A-IV with high density lipoprotein particles, serum high density lipoproteins were precipitated by incubating human serum with anti-apolipoprotein A-I immunoglobulins. At optimal concentrations, inducing a precipitation of 90 to 95% of serum apolipoprotein A-I, about 70% of serum apolipoprotein A-IV was precipitated. It was concluded that, in fasting human serum, apolipoprotein A-IV was mainly associated with high density lipoprotein particles. This high degree of association to high density lipoproteins did not result from the known in vitro redistribution of apolipoprotein A-IV induced by lecithin: cholesterol acyltransferase activity since it was observed in sera in the presence of inhibitors of this enzyme. The comparison of gel filtration profiles of total serum and of serum fractions separated by ultracentrifugation showed that the apolipoprotein A-IV-high density lipoprotein association was a weak one, easily dissociated by the ultracentrifugation process. The existence in fasting human serum of a predominant high density lipoprotein-associated form of apolipoprotein A-IV should stimulate more studies of the general function and metabolism of this protein.     

Interaction of apolipoprotein A-I in three different conformations with palmitoyl oleoyl phosphatidylcholine vesicles

Interactions of apolipoprotein A-I (apoA-I) with cell membranes appear to be important in the initial steps of reverse cholesterol transport. The objective of this work was to examine the effect of three distinct conformations of apoA-I (lipid-free and in 78 A or 96 A reconstituted high density lipoproteins, rHDL) on its ability to bind to, and abstract lipids from, palmitoyl oleoyl phosphatidylcholine membrane vesicles (small unilamellar vesicles, SUV, and giant unilamellar vesicles, GUV). The molecular interactions were observed by two-photon fluorescence microscopy, and the binding parameters were quantified by gel-permeation chromatography or isothermal titration microcalorimetry. Rearrangement of apoA-I-containing particles after exposure to SUVs was examined by native gel electrophoresis. The results indicate that lipid-free apoA-I binds reversibly, with high affinity, to the vesicles but does not abstract a significant amount of lipid nor perturb the vesicle structure. The 96 A rHDL, where all the amphipathic helices of apoA-I are saturated with lipid within the particles, do not bind to vesicles or perturb their structure. In contrast, the 78 A rHDL have a region of apoA-I, corresponding to a few amphipathic helical segments, which is available for external or internal phospholipid binding. These particles bind to vesicles with measurable affinity (lower than lipid-free apoA-I), abstract lipids from the membranes, and form particles of larger diameters, including 96 A rHDL. We conclude that the conformation of apoA-I regulates its binding affinity for phospholipid membranes and its ability to abstract lipids from the membranes.     

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...     

Characterization of lipoprotein particles isolated from the Golgi apparatus of rat liver

It has been proposed that particles within tubules and vesicles of the Golgi apparatus of liver cells are precursors of very low density lipoproteins in blood plasma. To characterize these particles we isolated a cell fraction rich in Golgi apparatus and associated particles from rat liver in quantities sufficient for analysis. Particles freed from the membranes of the Golgi apparatus and floated at d = 1.006 were studied by chemical analysis, immunodiffusion, and paper electrophoresis. The lipid composition of the Golgi particles was similar to that of very low density lipoproteins from the same rats. The protein content was about 10% of dry weight for both the Golgi particles and plasma very low density lipoproteins. The Golgi particles formed lines of identity with plasma very low density lipoproteins during immunodiffusion against antiserum to plasma very low density lipoproteins. On paper electrophoresis, however, many Golgi particles remained near the origin, with only a few migrating to the pre-beta position. It was concluded that the lipoproteins in the Golgi apparatus are the precursors of plasma very low density lipoproteins.