Transformation of large discoidal complexes of apolipoprotein A-I and phosphatidylcholine by lecithin-cholesterol acyltransferase

Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid.     

Reconstitution of apolipoprotein A-I from human high density lipoprotein with bovine brain sphingomyelin

Reconstitution of apolipoprotein A-I was found to occur readily with bovine brain sphingomyelin (BBSM), with a maximum rate occurring at a temperature of 28 degrees C, a temperature approximating the phase transition temperature for this naturally occurring phospholipid. At BBSM:A-I weight ratios of 7.5:1 or less, a single recombinant product was observed which contained three A-I molecules per particle, which had a BBSM:A-I molar ratio of 360 to 1 and which appeared in the electron microscope as a discoidal complex with a thickness of 68 A and a diameter of 217 A. By these criteria, as well as by gel filtration, this product appears very similar to that obtained by recombination of A-I with phosphatidylcholine at elevated ratios of phospholipid/protein. No evidence was found for the existence of any BBSM:A-I complexes comparable to the smaller lecithin:A-I complex containing 200-250 mol of phospholipid and two A-I molecules per complex which has been previously reported. At BBSM:A-I ratios of 15:1 (w/w), a new type of complex was observed which was discoidal by electron microscopy but possessed a larger diameter (390 A) and higher phospholipid:protein molar ratio (535:1) than has been observed previously for recombinant complexes. The BBSM:A-I complexes were found to be significantly more resistant to denaturation by guanidine hydrochloride than the dimyristoyl phosphatidylcholine:A-I recombinant complexes. It is concluded that the mechanisms of interaction between apolipoprotein A-I and either bovine brain sphingomyelin or phosphatidylcholines are similar, but that the nature of the protein-lipid interactions with BBSM are such as to produce larger and more stable complexes than are observed with the phosphatidylcholines.     

Molecular pathways in the transformation of model discoidal lipoprotein complexes induced by lecithin:cholesterol acyltransferase

Incubation (24 h, 37 degrees C) of discoidal complexes of phosphatidylcholine and apolipoprotein A-I (molar ratio 95 +/- 10 egg yolk phosphatidylcholine-apolipoprotein A-I; 10.5 X 4.0 nm, long X short dimension; designated, class 3 complexes) with the ultracentrifugal d greater than 1.21 g/ml fraction transformed the discoidal complexes to a small product with apparent mean hydrated and nonhydrated diameter of 7.8 and 6.6 nm, respectively. Formation of the small product was associated with marked reduction in phosphatidylcholine-apolipoprotein AI molar ratio of the complexes (on average from 95:1 to 45:1). Phospholipase A2 activity of lecithin:cholesterol acyltransferase participated in the depletion process, as evidenced by production of unesterified fatty acids. In the presence of the d greater than 1.21 g/ml fraction or partially purified lecithin:cholesterol acyltransferase and a source of unesterified cholesterol, the small product could be transformed to a core-containing (cholesteryl ester) round product with a hydrated and nonhydrated diameter of 8.6 and 7.5 nm, respectively. By means of cross-linking with dimethylsuberimidate, the protein moiety of the small product was shown to contain primarily two apolipoprotein A-I molecules per particle, while the large product contained three apolipoprotein A-I molecules per particle. The increase in number of apolipoprotein A-I molecules per particle during transformation of the small to the large product appeared to result from fusion of the small particles during core build-up and release of excess apolipoprotein A-I from the fusion product. The results obtained with the model complexes were consistent for the most part with recent observations (Chen, C., Applegate, K., King, W.C., Glomset, J.A., Norum, K.R. and Gjone, E. (1984) J. Lipid Res. 25, 269-282) on the transformation, by lecithin:cholesterol acyltransferase, of the small spherical high-density lipoproteins of patients with familial lecithin:cholesterol acyltransferase deficiency.     

Interaction of human-plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine, and characterization of the interaction products

Interaction of human low-density lipoproteins (LDL) with discoidal complexes comprised of egg yolk phosphatidylcholine and human apolipoprotein A-I (molar ratio, 88:1, respectively) was investigated. The multicomponent gradient gel electrophoretic pattern of LDL is transformed to one that includes a predominant component with an apparent particle diameter larger than that of the initial major LDL but still in the size range of normal LDL. The apparent particle diameter increase (range, 0.2-3.5 nm) is proportional to the increase (range, 6-40%) in LDL phospholipid/protein weight ratio following incubation (37 degrees C; 6 and 24 h); the smaller the initial LDL diameter, the greater the apparent particle diameter increase and percentage of phospholipid uptake. The LDL unesterified cholesterol/protein weight ratio decreases (range, 33-39%), but does not correlate with the increase in apparent particle diameter value. Interaction products are round particles with intact apolipoprotein B and show no evidence of phospholipid degradation. The products appear more dense than expected from the size vs. density relationship observed for nonincubated LDL subspecies. In addition to products in the normal LDL size range, larger components (apparent particle diameter range, 29.0-41.2 nm) also form and may be association complexes of phospholipid-modified LDL. Our results indicate that phospholipid uptake by LDL may contribute to the particle size polydispersity observed in plasma LDL.     

Structural studies of apolipoprotein A-I/phosphatidylcholine recombinants by high-field proton NMR, nondenaturing gradient gel electrophoresis, and electron microscopy

Complexes formed between apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) or egg phosphatidylcholine have been studied by high-field 1H NMR, nondenaturing gradient gel electrophoresis, electron microscopy, and gel filtration chromatography. Emphasis has been placed on an analysis of the particle size distribution within the micellar complexes produced at lipid/protein molar ratios of 40-700. As determined by electron microscopy and gel filtration of DMPC/apo A-I complexes, the size of the discoidal micelles produced appears to increase uniformly with an increasing lipid/protein ratio. By electron microscopy, the diameters of isolated DMPC/apo A-I discoidal micelles range from approximately 89 A at a 40 molar ratio to 205 A at a 700 molar ratio. Analysis of the micellar complexes by 1H NMR shows that concomitant with the increase in size is the progressive downfield shift of the choline N-methyl proton resonance of the complex which is observed from 3.245 to 3.267 ppm over the above molar ratio range. The relationship between chemical shift and micelle size is most simply interpreted as arising from a weighted averaging of two lipid environments--lipid-lipid and lipid-protein. In contrast to the above interpretation of the gel filtration experiments on DMPC/apo A-I complexes, nondenaturing gradient gel electrophoresis analysis of particle size distribution leads to an unexpected observation: as the DMPC/apo A-I ratio increases, discrete complexes of increasing size are formed in an apparently quantized manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of discoidal complexes of human apolipoprotein A-I with phosphatidylcholines containing various fatty acid chains

In this study we demonstrate that apolipoprotein A-I determined the common size classes of discoidal particles formed with numerous phosphatidylcholines, and with ether analogs of phosphatidylcholines. We show furthermore, that the nature of the lipids dictates the distribution of particles among the different size classes. These experiments were performed with discoidal complexes containing various phospholipids (phosphatidylcholines with saturated and unsaturated fatty acid chains of different lengths and the ether analog of 1-palmitoyl-2-oleoylphosphatidylcholine), cholesterol, and human apolipoprotein A-I, prepared by the sodium cholate dialysis method, and fractionated by Bio-Gel A-5m gel-filtration chromatography. The complex preparations were analyzed in terms of their average composition, spectral properties of the apolipoprotein, and the dynamic behavior of the lipid domains. Nondenaturing gradient gel electrophoresis was used to analyze the size classes of particles present in the complex preparations. Starting with reaction mixtures containing around 100:1, phospholipid/apolipoprotein A-I molar ratios, complexes were isolated with molar ratios from 40:1 to 100:1. In most complexes apolipoprotein A-I had high levels of alpha-helical structure (65-77% alpha-helix), and tryptophan residues in a nonpolar environment. The lipid domains of complexes exhibited the dynamic behavior expected of the main phospholipid components. In the average size range from 90 to 100 A diameters, discrete particle classes with 80, 87, 102, 108, or 112 A Stokes diameters were observed for all the complexes containing different phospholipids. These discrete, recurring particle sizes are attributed to distinct apolipoprotein A-I conformations and variable lipid content.     

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Reaction of lecithin: cholesterol acyltransferase with micellar substrates. Effect of particle sizes

Micellar, discoidal complexes were prepared from L-alpha-dipalmitoylphosphatidylcholine (DPPC) or egg phosphatidylcholine (egg-PC), cholesterol, and human apolipoprotein A-I by the cholate dialysis method. Reaction mixtures containing from 70:7:1 to 500:50:1, PC/cholesterol/apolipoprotein A-I (mol/mol) were fractionated by gel-filtration into various complex fractions. The isolated DPPC complexes ranged in size from 103 to 380 A in diameter, and in composition from 70:7:1 to 470:45:1, PC/cholesterol/apolipoprotein A-I (mol/mol), respectively. In contrast, the isolated egg-PC complexes only ranged in size from 105 to 214 A in diameter, and in composition from 65:5:1 to 153:17:1, PC/cholesterol/apolipoprotein A-I (mol/mol), respectively. Measurements of fluorescence wavelength maxima and fluorescence polarization of tryptophan residues of apolipoprotein A-I, in both series of complexes, revealed uniform spectral properties for all the egg-PC containing complexes. The DPPC complexes, on the other hand, had maxima in the fluorescence parameters for complexes with diameters around 200 A. When reacted with purified human lecithin:cholesterol acyltransferase, either at constant apolipoprotein A-I or at constant lipid concentration, all egg-PC complexes had very similar reaction rates, but the DPPC complex series exhibited major differences in reactivity. Minima in reaction rates occurred for DPPC complexes around 200 A in diameter, and optimal rates were observed with the small discoidal complexes (110 A in diameter). These reaction rates correlate well with the apolipoprotein A-I fluorescence properties and indicate that the apolipoprotein structure, reflected at the interface with phosphatidylcholine, may be the most important factor in determining complex reactivity with lecithin:cholesterol acyltransferase.     

Lipid-poor apolipoprotein A-I in Hep G2 cells: formation of lipid-rich particles by incubation with dimyristoylphosphatidylcholine

Apolipoprotein A-I is a major secretory product of the human hepatoma cell line, Hep G2; approx. 70% of apolipoprotein A-I was separated from the medium as lipid-poor apolipoprotein A-I in the d greater than 1.21 g/ml fraction while 30% was associated with high-density lipoproteins (HDL) of d 1.063-1.21 g/ml. The lipid-poor apolipoprotein A-I contains 50% proapolipoprotein A-I which is similar to the isoform distribution in Hep G2 preformed HDL. We tested the ability of lipid-poor apolipoprotein A-I from Hep G2 to form complexes with dimyristoylphosphatidylcholine (DMPC) vesicles at DMPC/apolipoprotein A-I molar ratios of 100:1 and 300:1. Lipid-poor apolipoprotein A-I was recovered in complex form while at a 300:1 ratio, 68.8 +/- 6.3% was recovered. On electron microscopy, the former complexes were small discs 16.9 nm +/- 4.5 S.D. in diameter while the latter were larger discs 21.4 +/- 4.4 nm diameter. Non-denaturing gradient gel electrophoresis of complexes formed at a 100:1 ratio had a peak in the region corresponding to 9.64 +/- 0.08 nm; these particles possessed two apolipoprotein A-I molecules. At the higher ratio, 300:1, two distinct complexes were identifiable, one which banded in the 9.7 nm region and the other in the 16.9-18.7 nm region. The former particles contained two molecules of apolipoprotein A-I and the latter, three molecules. This study demonstrates that lipid-poor apolipoprotein A-I which is rich in more basic isoforms forms discrete lipoprotein complexes similar to those formed by mature apolipoprotein A-I. It is further suggested that, under the appropriate conditions, precursor or nascent HDL may be assembled extracellularly.     

Reaction of lecithin:cholesterol acyltransferase with micellar complexes of apolipoprotein A-I and phosphatidylcholine, containing variable amounts of cholesterol

In a continued investigation of lecithin:cholesterol acyltransferase reaction with micellar, discoidal complexes of phosphatidylcholine (PC) . cholesterol . apolipoprotein A-I (apo-A-I), we prepared well defined complexes with variable free cholesterol contents and examined their reactivity with purified enzyme. The complexes, prepared by the sodium cholate dialysis method, were fractionated into "small" and "large" classes by gel filtration of the reaction mixtures through a Bio-Gel A-5m column. The small complexes had egg-PC/cholesterol/apo-A-I molar ratios from 68:14:1 to 80:1:1, discoidal shapes with diameters around 114 (+/- 13) A and widths of 42 A by electron microscopy, and Stokes radii from 47 to 49 A corresponding to molecular weights near 2 X 10(5). The corresponding properties of the large complexes, isolated from samples with higher cholesterol contents, were egg-PC/cholesterol/apo-A-I molar ratios from 84:26:1 to 96:17:1, diameters of 161 (+/- 20) A, widths of 43 A, Stokes radii around 80 A, and estimated molecular weights in the vicinity of 5 X 10(5). Both types of complexes, when adjusted to equal apo-A-I concentrations, gave essentially identical initial reaction velocities with purified lecithin:cholesterol acyltransferase over a wide range of cholesterol concentrations (from 2 X 10(-7) to 4 X 10(-4) M), PC/cholesterol molar ratios (from 3:1 to 12:1), and quite different lipid fluidity conditions as detected by diphenylhexatriene fluorescence polarization. When complexes were adjusted to a constant cholesterol concentration, the initial velocities of the lecithin:cholesterol acyltransferase reaction followed Michaelis-Menten kinetics relative to the apo-A-I concentrations. Arrhenius plots of initial reaction rates for various complexes with variable cholesterol content and fluidity, measured at constant apo-A-I concentrations, gave identical temperature dependences with an average activation energy of 18.0 kcal/mol. These results strongly suggest that the cholesterol esterification on high density lipoprotein particles does not depend on their unesterified-cholesterol contents, PC/unesterified-cholesterol molar ratios, nor on the fluidity of their lipid domains.     

Pathways in the formation of human plasma high density lipoprotein subpopulations containing apolipoprotein A-I without apolipoprotein A-II

The lecithin:cholesterol acyltransferase (LCAT)-induced transformation of two discrete species of model complexes that differ in number of apolipoprotein A-I (apoA-I) molecules per particle was investigated. One complex species (designated 3A-I(UC)-complexes) contained 3 apoA-I per particle, was discoidal (13.5 X 4.4 nm), and had a molar composition of 22:78:1 (unesterified cholesterol (UC):egg yolk phosphatidylcholine (egg yolk PC):apoA-I). The other complex species (designated 2A-I(UC)complexes) containing 2 apoA-I per particle was also discoidal (8.4 X 4.1 nm) and had a molar composition of 6:40:1. Transformation of 3A-I(UC)complexes by partially purified LCAT yielded a product (24 hr, 37 degrees C) with a cholesteryl ester (CE) core, 3 apoA-I, and a mean diameter of 9.2 nm. The 2A-I(UC)complexes were only partially transformed to a core-containing product (24 hr, 37 degrees C) which also had 3 apoA-I; this product, however, was smaller (diameter of 8.5 nm) than the product from 3A-I(UC)complexes. Transformation of 3A-I(UC)complexes appeared to result from build-up of core CE directly within the precursor complex. Transformation of 2A-I(UC)complexes, however, followed a stepwise pathway to the product with 3 apoA-I, apparently involving fusion of transforming precursors and release of one apoA-I from the fusion product. In the presence of low density lipoprotein (LDL), used as a source of additional cholesterol, conversion of 2A-I(UC)complexes to the product with 3 apoA-I was more extensive. The transformation product of 3A-I(UC)complexes in the presence of LDL also had 3 apoA-I but was considerably smaller in size (8.6 vs. 9.2 nm, diameter) and had a twofold lower molar content of PC compared with the product formed without LDL. LDL appeared to act both as a donor of UC and an acceptor of PC. Transformation products with 3 apoA-I obtained under the various experimental conditions in the present studies appear to be constrained in core CE content (between 13 to 22 CE per apoA-I; range of 9 CE molecules) but relatively flexible in content of surface PC molecules they can accommodate (between 24 to 49 PC per apoA-I; range of 25 PC molecules). The properties of the core-containing products with 3 apoA-I compare closely with those of the major subpopulation of human plasma HDL in the size range of 8.2-8.8 nm that contains the molecular weight equivalent of 3 apoA-I molecules.     

Characterization of complexes of egg yolk phosphatidylcholine and apolipoprotein A-II prepared in the absence and presence of sodium cholate

Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).     

Factors influencing interaction of human plasma low-density lipoproteins with discoidal complexes of apolipoprotein A-I and phosphatidylcholine

The effect of plasma components on the particle size distribution and chemical composition of human plasma low-density lipoproteins (LDL) during interaction with discoidal complexes of human apolipoprotein A-I and phosphatidylcholine (PC) was investigated. Incubation (37 degrees C, 1 h and 6 h) of LDL with discoidal complexes in the presence of the plasma ultracentrifugal d greater than 1.20 g/ml fraction (activity of lecithin-cholesterol acyltransferase inhibited) produces an increase in LDL apparent particle diameter two-to six-fold greater than that observed in the absence of the plasma d greater than 1.20 g/ml fraction. In incubation mixtures of LDL and discoidal complexes, both in the presence and absence of the plasma d greater than 1.20 g/ml fraction, the extent of LDL apparent particle diameter increase is: (1) approximately three-fold greater at 6 h than at 1 h, and (2) markedly greater for LDL with initially small (22.4-24.0 nm) major components than for LDL with initially large (26.2-26.8 nm) major components. The facilitation factor in the plasma d greater than 1.20 g/ml fraction is not plasma phospholipid transfer protein. Purified human serum albumin produces an apparent particle diameter increase comparable to the plasma d greater than 1.20 g/ml fraction. The discoidal complex-induced increase in LDL apparent particle diameter value by albumin is associated with an increase in phospholipid uptake by LDL and a decreased loss of LDL unesterified cholesterol. In preliminary experiments, high-density lipoproteins (HDL) reverse the apparent particle diameter increase originally induced by discoidal complexes. The presence of HDL (HDL phospholipid/LDL phospholipid molar ratio of 10:1) in the incubation (6 h) mixture of LDL and discoidal complexes also attenuates LDL apparent particle diameter increase. In vivo, the plasma LDL/HDL ratio may be a controlling factor in determining the extent to which phospholipid uptake and the associated change in LDL particle size distribution occurs.     

Isolation and lipid-binding properties of rat apolipoprotein A-IV

Apolipoprotein A-IV was isolated from the d less than 1.21 g/ml fraction of rat serum by gel filtration followed by heparin-Sepharose affinity chromatography; this method also facilitated the preparation of apolipoprotein A-I and apolipoprotein E. The apolipoprotein A-IV preparation was characterized by SDS-gel electrophoresis, isoelectric focusing, amino acid analysis and immunodiffusion. The lipid-binding properties of this protein were studied. Apolipoprotein A-IV associated with dimyristoylphosphatidylcholine (DMPC) to form recombinants which contained two molecules of apolipoprotein A-IV and had a lipid/protein molar ratio of 110. The density of the DMPC/apolipoprotein A-IV particles was determined to be 1.08 g/ml and the particles were visualized by electron microscopy as discs which were 5.8 nm thick and 18.0 nm in diameter. The stability of the DMPC/apolipoprotein A-IV recombinants, as determined by resistance to denaturation, was comparable to the stability of DMPC/apolipoprotein A-I complexes. However, by competition studies it was found that apolipoprotein A-I competed for the binding to DMPC more effectively than did apolipoprotein A-IV. It is concluded that, while rat apolipoprotein A-IV resembles other apolipoproteins in its lipid-binding characteristics, it may be displaced from lipid complexes by apolipoprotein A-I.     

High density lipoprotein particle size distribution in subjects with obstructive jaundice

High density lipoproteins (HDL) from 14 patients with obstructive jaundice were examined by gradient gel electrophoresis to determine the effect of obstruction on particle size distribution. HDL from 7 of these patients were fractionated by gel permeation chromatography and further characterized by electron microscopy, SDS gel electrophoresis, apolipoprotein A-I and apolipoprotein A-II immunoturbidimetry, and analysis of chemical composition. In addition, lecithin:cholesterol acyltransferase (LCAT) activity was measured and correlated with plasma apolipoprotein A-I concentration and particle size distribution. HDL were abnormal in all patients regardless of severity, cause, or duration of obstruction. The major HDL subfraction in normal subjects, HDL3a (radius 4.1-4.3 nm) was either absent or considerably diminished, and HDL2b (radius 5.3 nm) was also frequently absent. Very small particles comparable in size to normal HDL3c (radius 3.8 nm) were prominent. In patients with a bilirubin concentration greater than 250 mumol/l, normal HDL had totally disappeared and were replaced by large discoidal particles of radius 8.5 nm and small spherical particles of radius 3.6-3.7 nm. Both populations of particles were markedly depleted of cholesteryl ester and enriched in free cholesterol and phospholipid. The discoidal particles were rich in apolipoproteins E, A-I, A-II, and C, while the small spherical particles contained predominantly apolipoprotein A-I. LCAT activity was diminished in all subjects to 8-54% of normal, and was strongly positively correlated (r = 0.91 P less than 0.05) with plasma apolipoprotein A-I levels.     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

Recombinant models of lipoproteins. Apolipoprotein A-I/phosphatidylcholine/cholesterol complexes formed in a 2-chloroethanol-water mixture

Apolipoprotein A-I can spontaneously associate with phosphatidylcholine and cholesterol in 2-chloroethanol-water mixture. It was demonstrated, using a spin label technique, that dissolved molecules participate in complex formation. The apolipoprotein A-I/phosphatidylcholine/cholesterol complexes were isolated by gel chromatography. Complexes of three types were prepared and characterized: type A, large heterogeneous aggregates with molecular weight 600 000, sedimentation coefficient 10 S and the following molar composition - protein/phosphatidylcholine/cholesterol, 1:(70-100):(10-12); types B and C, with weight average molecular weights 140 000 and 110 000, average sedimentation coefficients 3.6 S and 1.7 S, respectively. Both types have the same molar composition - protein/phosphatidylcholine/cholesterol, 1:25:8. The dissimilar sedimentation coefficients between complexes B and C may be explained by the difference in the monomer/tetramer ratio (monomer molecular weight 50 000). The spin label sn-1-O-stearoyl-2-O-9'-spiro(4',4'-dimethyloxazolidine-3'-oxyl) heptadecanoylglycero-3-phosphocholine introduced into the complexes A and B showed different thermal properties of these complexes, which may be due to differences in the lipid-protein interactions.     

Formation of phospholipid-rich HDL: a model for square-packing lipoprotein particles found in interstitial fluid and in abetalipoproteinemic plasma

The major bovine HDL subfraction, fraction I-HDL, was incubated with increasing amounts of dimyristoylphosphatidylcholine (DMPC). HDL size, as determined by gradient gel electrophoresis and electron microscopy, increased with increasing HDL-phospholipid to DMPC mole ratios. Control fraction I-HDL were spherical, hexagonally-packing particles with a peak on gradient gel electrophoresis at 12.3 +/- 0.1 nm; at a ratio of 1:0.5, larger, mainly spherical particles with a peak at 12.9 +/- 0.08 nm were formed. At a ratio of 1:1, occasional square-shaped particles were seen by electron microscopy; by gradient gel analysis, the mean diameter of the HDL-product increased to 13.7 +/- 0.1 nm. At the 1:2 ratio, extensive domains of square-packing particles were noted; the major size peak of this product was 14.6 +/- 0.08 nm. In all incubations with DMPC, a small 9.4 +/- 0.08 nm product was formed; it was most pronounced at the 1:2 ratio. The large, less dense particles generated by incubation contained apolipoprotein A-I and small molecular weight proteins. The 9.4 nm product contained only apolipoprotein A-I. The less dense product formed during incubation at the 1:2 ratio had a decreased protein-to-lipid ratio relative to control HDL and a 2-fold increase in percent phospholipid. At a 1:2 ratio, incorporation of DMPC into fraction I-HDL results in the loss of one molecule of apolipoprotein A-I; the resultant particle is a stable phospholipid-rich and protein-poor HDL which has a square-packing geometry. These phospholipid-laden HDL are morphologically similar to lipoproteins isolated from interstitial fluid or from plasma of abetalipoproteinemic patients. Our data suggest that the unusual morphological properties of the latter biologically formed particles may be due to increases in the polar lipid contents, and concomitant decreases in surface protein.     

Interaction of model discoidal complexes of phosphatidylcholine and apolipoprotein A-I with plasma components. Physical and chemical properties of the transformed complexes

Conversion of model discoidal complexes of egg yolk phosphatidylcholine and apolipoprotein A-I, upon interaction with a source of lecithin:cholesterol acyltransferase (plasma d greater than or equal to 1.21 g/ml fraction or partially purified enzyme) and with different sources of substrate unesterified cholesterol (LDL, VLDL or cholesterol incorporated into complexes), was investigated by gradient gel electrophoresis, gel filtration, equilibrium density gradient ultracentrifugation, electron microscopy and chemical analysis. When the incubation mixture contained an inhibitor of lecithin:cholesterol acyltransferase, discoidal complexes with mean long dimension of approximately 10.5 +/- 1.9 nm were converted (within 1 h) predominantly to small round particles and were partially depleted of their phospholipid content. Upon electrophoresis the small particles showed peak maxima within the migration intervals of the human plasma ( HDL3b ) gge and ( HDL3c ) gge subpopulations with associated particle size ranges of 7.8-8.2 and 7.2-7.8 nm, respectively. Within 1 h, in the presence of activated enzyme, the complexes were again converted in major part to the small particles. However, further incubation resulted in an apparent single-step conversion to a larger major product with peak maximum occurring within the migration intervals of the ( HDL2a ) gge and the ( HDL3a ) gge subpopulations (particle size ranges 8.8-9.8 and 8.2-8.8 nm, respectively). Formation of an apolar core was indicated by detection of cholesteryl esters in the conversion product. The form in which the substrate unesterified cholesterol was introduced did not markedly influence the size properties of the final conversion product. With VLDL as source of substrate, considerable incorporation of triacylglycerol occurred in company with a lower level of cholesteryl esters, suggesting transfer of these lipids during formation of the apolar core. Incubation of complexes with a partially purified (3000-fold) preparation of lecithin:cholesterol acyltransferase yielded a product similar in properties to that when the d greater than or equal to 1.21 g/ml fraction was used. Our model discoidal complexes and their conversion products exhibit properties very similar to those of potential precursors to HDL as well as of mature HDL particles. Their further investigation shows promise of providing detailed insight into the possible origin and heterogeneity of human plasma HDL.     

Isolation and partial characterization of the lipoprotein families A and A-I from high-density lipoproteins of human serum

Procedures for the isolation of two lipoprotein fractions from plasma high-density lipoproteins (HDL), characterized by apolipoprotein A-I and apolipoprotein A-I together with apolipoprotein A-II, have been elaborated. Apolipoprotein A-I was identified as the protein moiety of one of these fractions (lipoprotein A-I) with polyacrylamide gel electrophoresis (at basic and acidic pH, as well as in the presence of sodium dodecyl sulphate), immuno-double-diffusion, and amino acid analysis. Apolipoproteins A-I and A-II were identified as the protein moiety of the other fraction (lipoprotein A) with polyacrylamide gel electrophoresis (basic and acidic pH) and immuno-double-diffusion. Lipoprotein A-I consisted of spherical particles with a diameter similar to that of HDL as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 8.7 nm from gel chromatography. Lipoprotein A-I migrated in the HDL position on crossed immunoelectrophoresis. On iso-electric focusing lipoprotein A-I appeared as multiple bands in the pH range 5.05-5.55. Lipoprotein A-I had the density of an HDL-2 fraction (rho: 1.063-1.105). Lipoprotein A consisted of spherical particles with a diameter similar to that of HDL, as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 7.9 nm from gel chromatography. The molar ratio between the A-I and A-II polypeptides was estimated to 1.3:1 with electroimmunoassay and calculations from the amino acid compositions. Lipoprotein A migrated in the position of HDL on crossed immuno-electrophoresis. On iso-electric focusing lipoprotein A appeared as one major and two minor bands in the pH range 5.10-5.30. Lipoprotein A had the hydrated density of an HDL-2 fraction.