Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.     

Defined apolipoprotein A-I conformations in reconstituted high density lipoprotein discs

We prepared and isolated defined, reconstituted high density lipoprotein (r-HDL) particles containing apolipoprotein A-I (apoA-I), palmitoyloleoylphosphatidylcholine, and cholesterol. The initial r-HDL were prepared by the sodium cholate method, then part of the preparation was depleted of phospholipid by exposure to LDL, and the resulting, stable r-HDL species were isolated by gel filtration. The isolated r-HDL were characterized in terms of their size, alpha-helix content, and the conformation of apoA-I as reported by the fluorescence properties of the tryptophan residues. Then the relative reactivity of the r-HDL with lecithin cholesterol acyltransferase was assessed. The isolated, discoidal r-HDL contained 2 and 3 apoA-I molecules/particle, and had 77 and 109 A diameters, respectively. Their spectral properties were essentially identical and were distinct from the larger particles in the class of r-HDL with 2 apoA-I molecules/particle (particles with diameters of 86 and 96 A). In addition, the reactivity of the 77 and 109 A particles with pure lecithin cholesterol acyltransferase was similar and about 10-fold lower than for the 86 and 96 A particles. We conclude that the stable, limiting r-HDL particles in each class (77 and 109 A) can arise from the larger particles of the same class by depletion of phospholipids. These limiting particles have very similar apoA-I conformations, with decreased alpha-helix contents and compact protein regions, that are very poor in activating lecithin cholesterol acyltransferase. Based on these results, we propose a model to explain the origin of the different classes and subclasses of the discoidal r-HDL particles.     

Synthesis of recombinant high density lipoprotein with apolipoprotein A-I and apolipoprotein A-V

It has been shown that apolipoprotein A-V (apoA-V) over-expression significantly lowers plasma triglyceride levels and decreases atherosclerotic lesion development. To assess the feasibility of recombinant high density lipoprotein (rHDL) reconstituted with apoA-V and apolipoprotein A-I (apoA-I) as a therapeutic agent for hyperlipidemic disorder and atherosclerosis, a series of rHDL were synthesized in vitro with various mass ratios of recombinant apoA-I and apoA-V. It is interesting to find that apoA-V of rHDL had no effect on lipoprotein lipase (LPL) activation in vitro and very low density lipoprotein (VLDL) clearance in HepG2 cells and in vivo. By contrast, LPL activation and VLDL clearance were inhibited by the addition of apoA-V to rHDL. Furthermore, the apoA-V of rHDL could not redistribute from rHDL to VLDL after incubation at 37°C for 30 min. These findings suggest that an increase of apoA-V in rHDL could not play a role in VLDL clearance in vitro and in vivo, which could, at least in part, attribute to the lost redistribution of apoA-V from rHDL to VLDL and LPL binding ability of apoA-V in rHDL. The therapeutic application of rHDL reconstituted with apoA-V and apoA-I might need the construction of rHDL from which apoA-V could freely redistribute to VLDL.     

Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency

Our aim was to characterize HDL subspecies and fat-soluble vitamin levels in a kindred with familial apolipoprotein A-I (apoA-I) deficiency. Sequencing of the APOA1 gene revealed a nonsense mutation at codon -2, Q[-2]X, with two documented homozygotes, eight heterozygotes, and two normal subjects in the kindred. Homozygotes presented markedly decreased HDL cholesterol levels, undetectable plasma apoA-1, tuboeruptive and planar xanthomas, mild corneal arcus and opacification, and severe premature coronary artery disease. In both homozygotes, analysis of HDL particles by two-dimensional gel electrophoresis revealed undetectable apoA-I, decreased amounts of small alpha-3 migrating apoA-II particles, and only modestly decreased normal amounts of slow alpha migrating apoA-IV- and apoE-containing HDL, while in the eight heterozygotes, there was loss of large alpha-1 HDL particles. There were no significant decreases in plasma fat-soluble vitamin levels noted in either homozygotes or heterozygotes compared with normal control subjects. Our data indicate that isolated apoA-I deficiency results in marked HDL deficiency with very low apoA-II alpha-3 HDL particles, modest reductions in the separate and distinct plasma apoA-IV and apoE HDL particles, tuboeruptive xanthomas, premature coronary atherosclerosis, and no evidence of fat malabsorption.     

Acid phosphatases bind to the main high density lipoprotein apolipoprotein A-I

Light-dependent Ca²⁺ efflux via the Ca²⁺/H⁺ antiport in the photosynthetic purple sulfur bacterium Chromatium vinosum was inhibited by three phenothiazines: chlorpromazine; trifluoperazine and phenothiazine. The inhibitors had no effect on Ca²⁺ uptake by C. vinosum in the dark nor any effect on the light-dependent efflux of either Na⁺ or Tl⁺ catalyzed, respectively, by the C. vinosum Na⁺/H⁺ or K⁺/H⁺ antiports. Ruthenium red and LaCl₃, neither of which inhibited light-dependent Ca²⁺ efflux in C. vinosum, markedly inhibited Ca²⁺ uptake in the dark by C. vinosum cells. Ruthenium red had no effect on the uptake of either Na⁺or the K⁺ analog T1⁺ by C. vinosum cells in the dark. These results have been interpreted in terms of two separate Ca²⁺ transport systems in C. vinosum: (i) a phenothiazine-sensitive and ruthenium red, La³⁺-insensitive Ca²⁺/H⁺ antiport responsible for Ca²⁺ efflux in the light; and (ii) a ruthenium red and La³⁺-sensitive but phenothiazine-insensitive Ca²⁺ uptake system.     

Cholesterol-induced alteration of apolipoprotein A-I conformation in reassembled high density lipoprotein.

Recent reports have shown that apolipoprotein A-I (apo A-I), the major protein of high density lipoprotein (HDL) may exist in different conformational states. We studied the effects of apolipoprotein A-II and/or cholesterol on the conformation of apo A-I in reassembled HDL. Analysis of tryptophan fluorescence quenching in the presence of iodine suggested that cholesterol increased the number of apo A-I tryptophan residues accessible to the aqueous phase, but decreased their mean degree of hydration. These observations cannot be totally explained on the basis of the effect of cholesterol on phospholipid viscosity as determined by fluorescence anisotropy of diphenyl hexatriene. We did not observe any effect of apo A-II on the conformation of apo A-I.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Oxidation of methionine residues affects the structure and stability of apolipoprotein A-I in reconstituted high density lipoprotein particles.

To determine the effect of oxidative damage to lipid-bound apolipoprotein A-I (apo A-I) on its structure and stability that might be related to previously observed functional disorders of oxidized apo A-I in high density lipoproteins (HDL), we prepared homogeneous reconstituted HDL (rHDL) particles containing unoxidized apo A-I and its commonly occurring oxidized form (Met-112, 148 bis-sulfoxide). The size of the obtained discoidal rHDL particles ranged from 9.0 to 10.0 nm and did not depend upon the content of the oxidized protein. Using circular dichroism methods, no change in the secondary structure of lipid-bound oxidized apo A-I was found. Isothermal and thermal denaturation experiments showed a significant destabilization of the oxidized protein to denaturation by guanidine hydrochloride or heat. This effect was observed with and without co-reconstituted apolipoprotein A-II. Limited tryptic digestion indicated that the central region of oxidatively damaged apo A-I becomes exposed to proteolysis in the rHDL particles. Implications of these data for apolipoprotein function are discussed.     

Structure of apolipoprotein A-I in three homogeneous, reconstituted high density lipoprotein particles

To elucidate further the conformation of human apolipoprotein A-I (apoA-I) in lipid-bound states and its effect on the reaction with lecithin cholesterol acyltransferase (LCAT), we prepared reconstituted HDL (rHDL) particles from a reaction mixture containing dipalmitoylphosphatidylcholine/cholesterol/apoA-I in the molar ratios of 150:7.5:1. The particles were separated by gel filtration into three classes of highly homogeneous and reproducible discs with diameters of 97, 136, and 186 A, containing 2, 3, and 4 molecules of apoA-I/disc, respectively, and increasing proportions of phospholipid and cholesterol. These three classes of particles were then investigated by a variety of fluorescence techniques, to probe the average environment and mobility of the tryptophan (Trp) residues in the structure of apoA-I. We found small, gradual changes in the fluorescence parameters with changes in the size of the rHDL, consistent with a shift of Trp residues to a more hydrophobic and more rigid environment, as well as an increased resistance of apoA-I to denaturation by guanidine hydrochloride in the larger particles. In contrast, circular dichroism measurements and binding studies with seven monoclonal antibodies indicated a similar alpha-helical structure (73%) for apoA-I in all the particles, and similar exposure of apoA-I epitopes in the COOH-terminal two-thirds of the apolipoprotein. Thus the structure of apoA-I is comparable for the three classes of particles and is consistent with the presence of eight alpha-helical segments per apoA-I in contact with the lipid. In addition, we obtained the apparent kinetic parameters for the reaction of the rHDL particles with lecithin cholesterol acyltransferase. The apparent Km values were similar but the apparent Vmax decreased almost 8-fold, going from the 97- to the 186-A particles; therefore, the decreasing reactivity for the larger particles can be attributed mainly to differences in the catalytic rate constant. The rate limiting step is probably affected by local structural differences in the apoA-I, or by the interfacial properties of the lipid.     

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.     

Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

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.     

Human apolipoprotein A-I forms small high density lipoprotein particles in rats in vivo.

The effects of injection of purified human or rat apolipoprotein (apo) A-I (1.7 mg/100 g body weight) on the size and composition of rat high density lipoprotein (HDL) particles have been investigated. The injection of human apo A-I results in the formation (over a period of 3 to 6 h) of a population of smaller HDL particles resembling human HDL3. This population of smaller particles contains human apo A-I and rat apo A-IV but lacks rat apo A-I and rat apo E. Small HDL3-like particles are not detected in rat plasma following the injection of rat apo A-I. Associated with the injection of either human or rat apo A-I is a gradual increase of plasma cholesterol levels of 20 to 50% (over 24 h) and the appearance of larger HDL particles. The results suggest that the smaller HDL particles in human plasma compared to rat plasma are not simply due to the action of lipid modifying enzymes or lipid transfer proteins but a specific property of human apo A-I.     

Prebeta high density lipoprotein has two metabolic fates in human apolipoprotein A-I transgenic mice

We compared the in vivo metabolism of prebeta HDL particles isolated by anti-human apolipoprotein A-I (apoA-I) immunoaffinity chromatography (LpA-I) in human apoA-I transgenic (hA-I Tg) mice with that of lipid-free apoA-I (LFA-I) and small LpA-I. After injection, prebeta LpA-I were removed from plasma more rapidly than were LFA-I and small LpA-I. Prebeta LpA-I and LFA-I were preferentially degraded by kidney compared with liver; small LpA-I were preferentially degraded by the liver. Five minutes after tracer injection, 99% of LFA-I in plasma was found to be associated with medium-sized (8.6 nm) HDL, whereas only 37% of prebeta tracer remodeled to medium-sized HDL. Injection of prebeta LpA-I doses into C57Bl/6 recipients resulted in a slower plasma decay compared with hA-I Tg recipients and a greater proportion (>60%) of the prebeta radiolabel that was associated with medium-sized HDL. Prebeta LpA-I contained one to four molecules of phosphatidylcholine per molecule of apoA-I, whereas LFA-I contained less than one. We conclude that prebeta LpA-I has two metabolic fates in vivo, rapid removal from plasma and catabolism by kidney or remodeling to medium-sized HDL, which we hypothesize is determined by the amount of lipid associated with the prebeta particle and the particle's ability to bind to medium-sized HDL.     

A detailed molecular belt model for apolipoprotein A-I in discoidal high density lipoprotein.

Apolipoprotein A-I (apoA-I) is the principal protein of high density lipoprotein particles (HDL). ApoA-I contains a globular N-terminal domain (residues 1-43) and a lipid-binding C-terminal domain (residues 44-243). Here we propose a detailed model for the smallest discoidal HDL, consisting of two apoA-I molecules wrapped beltwise around a small patch of bilayer containing 160 lipid molecules. The C-terminal domain of each monomer is ringlike, a curved, planar amphipathic alpha helix with an average of 3.67 residues per turn, and with the hydrophobic surface curved toward the lipids. We have explored all possible geometries for forming the dimer of stacked rings, subject to the hypothesis that the optimal geometry will maximize intermolecular salt bridge interactions. The resulting model is an antiparallel arrangement with an alignment matching that of the (nonplanar) crystal structure of lipid-free apoA-I.     

High level expression of human apolipoprotein A-I in transgenic rats raises total serum high density lipoprotein cholesterol and lowers rat apolipoprotein A-I

To examine the consequences of increased apolipoprotein A-I production on cholesterol and lipoprotein metabolism, we have produced two lines of transgenic rats; one expressing moderate and one very high levels of human apolipoprotein A-I. The rats were produced by microinjection of a 13 kbp DNA fragment containing the human apolipoprotein A-I gene plus 10 kbp of its 5′ flanking sequence and 1 kbp of its 3′ flanking sequence. Both lines of transgenic rats express human apolipoprotein A-I mRNA in liver and human apolipoprotein A-I in plasma. Sera from these rats contain significantly higher levels of total apolipoprotein A-I, high density lipoprotein cholesterol and phospholipid than sera from non-transgenic littermates. Transgenic rats expressing high levels of human apolipoprotein A-I have reduced levels of serum rat apolipoprotein A-I suggesting a mechanism exists to down-regulate apolipoprotein A-I production. These transgenic rats provide a unique animal model to examine the effects of increased apolipoprotein A-I production on lipid and lipoprotein metabolism.     

Dissociation of the lipid-enzyme complex of hormone-sensitive lipase using high density lipoprotein or apolipoprotein A-I

Hormone-sensitive lipase in homogenates of adipose tissue occurs as a large, lipid-rich complex including several acylhydrolase activities that emerge quantitatively in the void volume on gel filtration chromatography (2% agarose). Incubation with intact human plasma high density lipoprotein or with lipid-free apolipoprotein A-I, however, disrupted the lipid-rich complex almost completely and most of the enzyme activity eluted from a 2% agarose column at about Ve = 2.3 x Vo. This use of the detergent-like properties of apolipoprotein A-I may be of value for dissociation of other lipid-associated or membrane-bound enzymes.     

Measurement of apolipoprotein A-I in rat high density lipoprotein and in rat plasma by radioimmunoassay.

A double antibody radioimmunoassay (RIA) for rat apolipoprotein A-I is reported. The ApoA-I isolated from delipidated HDL by gel filtration yielded a single band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS), and its amino acid composition resembled that reported by others. ApoA-I was iodinated by lactoperoxidase and the resulting 125I-apoA-I was purified by gel filtration. Up to 93% of 125I-apoA-I was precipitable by antibody and greater than 99% of bound 125I-apoA-I was displaced by "cold" apoA-I. Other rat lopoproteins and apolipoproteins did not react in this system. Human plasma were also not reactive, nor were dog, goat, and sheep plasmas.     

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.