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.     

Apolipoprotein A-I assumes a "looped belt" conformation on reconstituted high density lipoprotein

Apolipoprotein A-I (apoA-I) plays a central role in the reverse cholesterol transport pathway; however, the structural basis for its antiatherogenic effects remains poorly understood. Here we employ EPR spectroscopy and fluorescence resonance energy transfer to elucidate the conformation and relative alignment of apoA-I monomers on discoidal (9.4 nm) reconstituted high density lipoprotein (rHDL). EPR spectroscopy provided evidence for an extended helical secondary structure. Position 139 since it was the only residue examined to display a dynamic motional character consistent with a flexible loop structure. The EPR spectra of nitroxide probes at positions 133 and 146 exhibit spin coupling, indicating that these positions are proximal to an apoA-I paired counterpart on the perimeter of rHDL. fluorescence resonance energy transfer studies employing engineered apoA-I variants possessing a single tryptophan (energy donor) and/or a single cysteine (whose thiol moiety was covalently labeled with an extrinsic energy acceptor) provided evidence that paired apoA-I molecules around the perimeter of rHDL align in an extended antiparallel conformation. Taken together with the observation that the EPR spectra of nitroxide probes positioned at intervening sequence positions (134-145) do not exhibit spin coupling, this has led us to propose a "looped belt" model, wherein residues 133-146 comprise a flexible loop segment that confers to apoA-I an intrinsic ability to adapt its structure to accommodate changing particle lipid content. Specifically, in the looped belt model, with the exception of amino acids 134-145, apoA-I aligns with its counterpart in a helix 5-helix 5 registry, centered at position 139.     

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

While low apolipoprotein A-I (apoA-I) levels are primarily associated with increased high density lipoprotein (HDL) fractional catabolic rate (FCR), the factors that regulate the clearance of HDL from the plasma are unclear. In this study, the effect of lipid composition of reconstituted HDL particles (LpA-I) on their rate of clearance from rabbit plasma has been investigated. Sonicated LpA-I containing 1 to 2 molecules of purified human apoA-I and 5 to 120 molecules of palmitoyl-oleoyl phosphatidylcholine (POPC) exhibit similar charge and plasma FCR to that for lipid free apoA-I, 2.8 pools/day. Inclusion of 1 molecule of apoA-II to an LpA-I complex increases the FCR to 3.5 pools/day, a value similar to that observed for exchanged-labeled HDL3. In contrast, addition of 40 molecules of triglyceride, diglyceride, or cholesteryl ester to a sonicated LpA-I containing 120 moles of POPC and 2 molecules of apoA-I increases the negative charge of the particle and reduces the FCR to 1.8 pools/day. Discoidal LpA-I are the most positively charged lipoprotein particles and also have the fastest clearance rates, 4.5 pools/day. Immunochemical characterization of the different LpA-I particles shows that the exposure of an epitope at residues 98 to 121 of the apoA-I molecule is associated with an increased negative particle charge and a slower clearance from the plasma.We conclude that the charge and conformation of apoA-I are sensitive to the lipid composition of LpA-I and play a central role in regulating the clearance of these lipoproteins from plasma. conformation regulate the clearance of reconstituted high density lipoprotein in vivo.     

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.     

Apolipoprotein A-I activates protein kinase C alpha signaling to phosphorylate and stabilize ATP binding cassette transporter A1 for the high density lipoprotein assembly

ATP-binding cassette transporter A1 (ABCA1) plays an essential role in the helical apolipoprotein-mediated assembly of high density lipoprotein, and the apolipoporteins stabilize ABCA1 against calpain-mediated degradation during the reaction ((2002) J. Biol. Chem. 277, 22426-22429). Protein kinase C (PKC) inhibitors suppressed both ABCA1 stabilization and cellular lipid release mediated by apolipoprotein A-I (apoA-I) but not ABCA1 increase by calpain inhibitors. The increase of ABCA1 and the cellular lipid release by apoA-I were both suppressed by a phosphatidylcholine phospholipase C (PC-PLC) inhibitor but not by the inhibitors of phosphatidylinositol-PLC and phosphatidylinositol 3-kinase. A protein phosphatase inhibitor further enhanced the ABCA1 increase by apoA-I. Biochemical and microscopic evidence indicated that apoA-I activated PKC alpha, and phosphorylation of ABCA1 was directly demonstrated by apoA-I via PKC. Finally, digestion of sphingomyelin increased ABCA1, and a PC-PLC inhibitor suppressed it. We conclude that apoA-I activates PKC alpha by PC-PLC-mediated generation of diacylglycerol initiated by the removal of cellular sphingomyelin ((2002) J. Biol. Chem. 277, 44709-44714), and subsequently phosphorylates and stabilizes ABCA1.     

Apolipoprotein A-I localization and dipalmitoylphosphatidylcholine dynamics in reconstituted high density lipoproteins

The structure and molecular dynamics of recombinant high density lipoproteins (rHDL) were studied by non-radiative energy transfer (NRET), fluorescence anisotropy and intensity measurements. The rHDL particles contained human plasma apolipoprotein (apo) A-I and dipalmitoylphosphatidylcholine (DPPC). Fluorescent cis- and trans-parinaric acids were used both as probes of molecular motion in the particle lipid phase and as acceptors in the Forster's energy transfer from apo A-I tryptophan residues to determine particle dimensions, apolipoprotein localization and lipid dynamics. The probes are sensitive to thermal wobbling (macromobility) and conformational deformations (micromobility) of phospholipid acyl chains. The experimental data fitted to various models of the particle structure are compatible with the following: (a) at T < Tt the particles appeared as lens-like discs with a radius of the lipid phase of 5 nm and a mean thickness of 4 nm, the value being more by 20% in the particle centre, the alpha-helices of about 1 nm thickness were located around the edge of the lipid core. Compared to liposomes, both macro- and micromobility of DPPC molecules in rHDL were more rapid due to a significant disorder of the boundary lipid molecules close to the apo A-I molecule. This disorder led to the increase of the specific surface area per one lipid molecule, S(o). The lipid phase can be divided into three regions: (i) zone I of the most tightly packed lipid (0-1.7 nm from the disc axis) with a S(o) value small as 0.5 nm2; (ii) intermediate zone II (from 1.7 to 4.0 nm); and (iii) boundary lipid zone III (4-5 nm) of significantly disordered lipid with a S(o) value large as 0.65 nm2. (b) at T> Tt the S(o) heterogeneity disappeared, the radius of the lipid phase did not increase significantly, not exceeding 5.2-5.4 nm, but protein-induced immobilization of lipid molecules which affected about half or more of the total lipid, became remarkable. The overall effect was the suppression of the transition amplitude in rHDL compared to liposomes. The structural inhomogeneity might underlie the function of the native plasma HDL as the key component of the transport and metabolism of plasma lipids.     

Apolipoprotein A-I adopts a belt-like orientation in reconstituted high density lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein associated with high density lipoprotein (HDL), and its plasma levels have been correlated with protection against atherosclerosis. Unfortunately, the structural basis of this phenomenon is not fully understood. Over 25 years of study have produced two general models of apoA-I structure in discoidal HDL complexes. The "belt" model states that the amphipathic helices of apoA-I are aligned perpendicular to the acyl chains of the lipid bilayer, whereas the "picket fence" model argues that the helices are aligned parallel with the acyl chains. To distinguish between the two models, various single tryptophan mutants of apoA-I were analyzed in reconstituted, discoidal HDL particles composed of phospholipids containing nitroxide spin labels at various positions along the acyl chain. We have previously used this technique to show that the orientation of helix 4 of apoA-I is most consistent with the belt model. In this study, we performed additional control experiments on helix 4, and we extended the results by performing the same analysis on the remaining 22-mer helices (helices 1, 2, 5, 6, 7, 8, and 10) of human apoA-I. For each helix, two different mutants were produced that each contained a probe Trp occurring two helical turns apart. In the belt model, the two Trp residues in each helix should exhibit maximal quenching at the same nitroxide group position on the lipid acyl chains. For the picket fence model, maximal quenching should occur at two different levels in the bilayer. The results show that the majority of the helices are in an orientation that is consistent with a belt model, because most Trp residues localized to a position about 5 A from the center of the bilayer. This study corroborates a belt hypothesis for the majority of the helices of apoA-I in phospholipid discs.     

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.     

Apolipoprotein E and lipoprotein lipase secreted from human monocyte-derived macrophages modulate very low density lipoprotein uptake

We investigated the roles of lipoprotein lipase and apolipoprotein E (apoE) secreted from human monocyte-derived macrophages in the uptake of very low density lipoproteins (VLDL). ApoCII-deficient VLDL were isolated from a patient with apoCII deficiency. The lipolytic conversion to higher density and the degradation of the apoCII-deficient VLDL by macrophages were very slight, whereas the addition of apoCII enhanced both their conversion and degradation. This suggests that the lipolysis and subsequent conversion of VLDL to lipoproteins of higher density are essential for the VLDL uptake by macrophages. VLDL incubated with macrophages obtained from subjects with E3/3 phenotype (E3/3-macrophages) showed a 17-fold greater affinity in inhibiting the binding of 2 micrograms/ml 125I-low density lipoprotein (LDL) to fibroblasts than native VLDL, whereas the incubation of VLDL with macrophages obtained from a subject with E2/2 phenotype (E2/2-macrophages) did not cause any increase in their affinity. Furthermore, 3 micrograms/ml 125I-VLDL obtained from a subject with E3/3 phenotype were degraded by E3/3-macrophages to a greater extent than by E2/2-macrophages (2-fold), indicating that VLDL uptake is influenced by the phenotype of apoE secreted by macrophages. From these results, we conclude that both lipolysis by lipoprotein lipase and incorporation of apoE secreted from macrophages alter the affinity of VLDL for the LDL receptors on the cells, resulting in facilitation of their receptor-mediated endocytosis.     

NFkappaB regulates plasma apolipoprotein A-I and high density lipoprotein cholesterol through inhibition of peroxisome proliferator-activated receptor alpha

The levels of plasma HDL cholesterol and apoA-I in NFkappaB p50 subunit-deficient mice were significantly higher than those in wild-type mice under regular and high fat diets, without any significant difference in the level of total cholesterol. To examine the role of NFkappaBin lipid metabolism, we studied its effect on the regulation of apoA-I secretion from human hepatoma HepG2 cells. Lipopolysaccharide-induced activation of NFkappaB reduced the expression of apoA-I mRNA and protein, whereas adenovirus-mediated expression of IkappaBalpha super-repressor ameliorated the reduction. This IkappaBalpha-induced apoA-I increase was blocked by preincubation with MK886, a selective inhibitor of peroxisome proliferator-activated receptor alpha (PPARalpha), suggesting that NFkappaB inactivation induces apoA-I through activation of PPARalpha. To further support this idea, the expression of IkappaBalpha increased apoA-I promoter activity, and this increase was blocked by preincubation with MK886. Mutations in the putative PPARalpha-binding site in the apoA-I promoter or lack of the site abrogated these changes. Taking these results together, inhibition of NFkappaB increases apoA-I and HDL cholesterol through activation of PPARalpha in vivo and in vitro. Our data suggest a new aspect of lipid metabolism and may lead to a new paradigm for prevention and treatment of atherosclerotic disease.     

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.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

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.     

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.     

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.     

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.     

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.     

Apolipoprotein A-I is necessary for the in vivo formation of high density lipoprotein competent for scavenger receptor BI-mediated cholesteryl ester-selective uptake

The severe depletion of cholesteryl ester (CE) in steroidogenic cells of apoA-I(-/-) mice suggests that apolipoprotein (apo) A-I plays a specific role in the high density lipoprotein (HDL) CE-selective uptake process mediated by scavenger receptor BI (SR-BI) in vivo. The nature of this role, however, is unclear because a variety of apolipoproteins bind to SR-BI expressed in transfected cells. In this study the role of apoA-I in SR-BI-mediated HDL CE-selective uptake was tested via analyses of the biochemical properties of apoA-I(-/-) HDL and its interaction with SR-BI on adrenocortical cells, hepatoma cells, and cells expressing a transfected SR-BI. apoA-I(-/-) HDL are large heterogeneous particles with a core consisting predominantly of CE and a surface enriched in phospholipid, free cholesterol, apoA-II, and apoE. Functional analysis showed apoA-I(-/-) HDL to bind to SR-BI with the same or higher affinity as compared with apoA-I(+/+) HDL, but apoA-I(-/-) HDL showed a 2-3-fold decrease in the V(max) for CE transfer from the HDL particle to adrenal cells. These results indicate that the absence of apoA-I results in HDL particles with a reduced capacity for SR-BI-mediated CE-selective uptake. The reduced V(max) illustrates that HDL properties necessary for binding to SR-BI are distinct from those properties necessary for the transfer of HDL CE from the core of the HDL particle to the plasma membrane. The reduced V(max) for HDL CE-selective uptake likely contributes to the severe reduction in CE accumulation in steroidogenic cells of apoA-I(-/-) mice.