In vivo metabolism of apolipoprotein A-I on high density lipoprotein particles LpA-I and LpA-I,A-II.

Apolipoprotein (apo) A-I is the major protein in high density lipoproteins (HDL) and is found in two major subclasses of lipoproteins, those containing apolipoprotein A-II (termed LpA-I,A-II) and those without apoA-II (termed LpA-I). The in vivo kinetics of apoA-I on LpA-I and LpA-I,A-II were investigated in normolipidemic human subjects. In the first series of studies, radiolabeled apoA-I and apoA-II were reassociated with autologous plasma lipoproteins and injected into normal subjects. LpA-I and LpA-I,A-II were isolated from plasma at selected time points by immunoaffinity chromatography. By 24 h after injection, only 52.8 +/- 1.0% of the apoA-I in LpA-I remained, whereas 66.9 +/- 2.7% of apoA-I in LpA-I,A-II remained (P less than 0.01). In the second series of studies, purified apoA-I was labeled with either 131I or 125I and reassociated with autologous plasma. Isolated LpA-I and LpA-I,A-II particles differentially labeled with 131I-labeled apoA-I and 125I-labeled apoA-I, respectively, were simultaneously injected into study subjects. The plasma residence time of apoA-I injected on LpA-I (mean 4.39 days) was substantially shorter than that of apoA-I injected on LpA-I,A-II (mean 5.17 days), with a mean difference in residence times of 0.79 +/- 0.08 days (P less than 0.001). These data demonstrate that apoA-I injected on LpA-I is catabolized more rapidly than apoA-I injected on LpA-I,A-II. The results are consistent with the concept that LpA-I and LpA-I,A-II have divergent metabolic pathways.     

High density lipoprotein metabolism in normolipidemic and cholesterol-fed rabbits.

New Zealand white rabbits were used to determine the compositional and metabolic changes induced in high density lipoproteins (HDL, rho = 1.063--1.21 g/ml) in response to cholesterol feeding. There was no change in total HDL cholesterol in plasma due to cholesterol feeding (12 weeks), but the triglyceride level was decreased to 29% of pretreatment values. Total protein content of HDL was decreased in response to cholesterol feeding, resulting in a significant increase in the cholesterol/protein ratio. There was a decrease in some isomer of the major apolipoproteins (A-I2) of HDL. The decay of radioactivity in HDL or its apolipoproteins was biphasic in both normolipidemic and hypercholesterolemic rabbits. The first phase was much more rapid than the second. The decay rates for the radioactivity in HDL depended upon the dietary status of the recipient animal.     

Free apolipoproteins A-I and A-IV present in human plasma displace high-density lipoprotein on cultured bovine aortic endothelial cells

Adult bovine aortic endothelial (ABAE) cells, exposed to serum-free medium, specifically bind 125I-labeled human high-density lipoprotein (125I-HDL). Addition of human lipoprotein-deficient serum (LPDS) reduces the specific binding of 125I-HDL in a concentration-dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I-HDL. ABAE cultures exposed to 125I-labeled LPDS (125I-LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I-LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25-hydroxycholesterol (25-HC) results in an increase in the binding of the two 125I-LPDS components, similar to the increase observed in 125I-HDL binding in the presence of 25-HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with apolipoproteins A-I and A-IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transferred from the SDS gel to nitrocellulose paper and interacted specifically with anti-(A-I) and anti-(A-IV) sera respectively. When ABAE cultures, pretreated with 25-HC in the presence of LPDS, are subjected to cell-surface iodination, the A-IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A-IV with the cell surface of 25-HC-treated cells is not specific to ABAE cells and appears also in human skin fibroblasts. Analysis of the relative amounts of various apolipoproteins in the 125I-HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A-II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I-HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I-HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A-I and A-IV in the 125I-HDL by unlabeled A-I and A-IV present in LPDS. The results indicate that free apolipoproteins A-I and A-IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A-I and A-IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL.     

Low density lipoprotein metabolism in hypertriglyceridemic and normolipidemic patients with coronary heart disease

The turnover rates of low density lipoprotein-apolipoprotein B (LDL-apoB) were determined in 32 men with coronary heart disease (CHD) and 11 control men with normal plasma lipids. Thirty patients with CHD had normal levels of LDL-cholesterol (LDL-C); of these patients, 9 had hypertriglyceridemia and 21 had normal plasma lipids. Mean concentrations of total cholesterol and LDL-C were similar among the control subjects and CHD patients, although the latter had significantly lower HDL-C. In control subjects, transport rates and fractional catabolic rates (FCR) of LDL-B were 10.6 +/- 0.5 (SEM) mg/kg-day and 0.31 +/- 0.01 pools/day, respectively. In 10 hypertriglyceridemic patients with CHD, transport rates were 21.7 +/- 1.7 mg/kg-day, and FCRs averaged 0.56 +/- 0.06 pools/day; both were significantly higher than normal (P less than 0.05). Six normolipidemic patients also had abnormally high transport rates of LDL-apoB (19.4 +/- 2.8 mg/kg-day) and FCRs (0.51 +/- 0.03 pools/day); again both were higher than normal. The remaining 16 normolipidemic patients with CHD had normal transport rates (9.9 +/- 0.6 mg/kg-day) and FCRs (0.28 +/- 0.01 pools/day). Thus, hypertriglyceridemic patients with CHD and a portion of normolipidemic patients with CHD were characterized by increases in both transport and fractional catabolic rate of LDL-apoB; these abnormalities in LDL metabolism may have contributed to their coronary heart disease. However, the majority of normolipidemic patients with CHD did not show a distinct defect in their LDL metabolism.     

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.     

Comparison of the metabolic behavior of rat apolipoproteins A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins

Rat apolipoprotein (apo) A-I and A-IV, isolated from both lymph chylomicrons and serum high density lipoproteins (HDL) were analyzed by isoelectric focusing. Lymph chylomicron apo A-I consisted for 81 +/- 2% of the pro form and for 19 +/- 2% of the mature form, while apo A-I isolated from serum HDL was present for 36 +/- 4% in the pro form and for 64 +/- 4% in the mature form. Apo A-IV also showed two major protein bands after analysis by isoelectric focusing. The most prominent component is the more basic protein that amounts to 80 +/- 2% in apo A-IV isolated from lymph chylomicrons and to 60 +/- 3% in apo A-IV isolated from serum HDL. Apo A-I (or apo A-IV), isolated from both sources (lymph chylomicrons or serum HDL), was iodinated and the radioactive apolipoproteins were incorporated into rat serum lipoproteins. The resulting labeled HDL was isolated from serum by molecular sieve chromatography on 6% agarose columns and injected intravenously into rats. No difference in the fractional turnover rate or the tissue uptake of the two labeled HDL preparations was observed, neither for apo A-I nor for apo A-IV. It is concluded that the physiological significance of the extracellular pro apo A-I conversion or the post-translational modification of apo A-IV is not related to the fractional turnover rate in serum or to the rate of catabolism in liver and kidneys.     

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 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.     

Chromatofocusing of apolipoproteins from human serum high density lipoprotein

Human HDL was delipidated and the apolipoproteins were fractionated by chromatofocusing. Chromatofocusing, which separates proteins due to their differing isoelectric points, resulted in 8 peaks with corresponding pI values of 7.40, 6.92, 6.64, 5.48, 5.30, 5.18, 4.92 and 4.63. By one single chromatofocusing run four apolipoproteins were obtained in pure form. Two additional polypeptides could be purified during the desalting step using phenyl-Sepharose.     

Transport of lipid and apolipoproteins A-I and A-IV in intestinal lymph of the rat

Intestinal lipid absorption is associated with marked increases in the synthesis and secretion of apolipoprotein A-IV (apoA-IV) by the small intestine. Whether the increased intestinal apoA-IV synthesis and secretion results from increased fat uptake, increased cellular triglyceride (TG) content, or increased secretion of TG-rich lipoproteins by the enterocytes is unknown. Previous work from this laboratory has shown that a hydrophobic surfactant, Pluronic L-81 (L-81), is a potent inhibitor of intestinal formation of chylomicrons (CM), without reducing fat uptake or re-synthesis to TG. Furthermore, this inhibition can be reversed quickly by the cessation of L-81 infusion. Thus L-81 offers a unique opportunity to study the relationship between lymphatic TG, apoA-I and A-IV secretion. In this study, we studied the lymphatic transport of TG, apoA-I, and apoA-IV during both the inhibitory phase (L-81 infused together with lipid) and the subsequent unblocking phase (saline infusion). Two groups of lymph fistula rats were used, the control and the experimental rats. In the experimental rats, a phosphate-buffered taurocholate-stabilized emulsion containing 40 mumol [3H]triolein, 7.8 mumol of phosphatidylcholine, and 1 mg L-81 per 3 ml was infused at 3 ml/h for 8 h. This was then replaced by glucose-saline infusion for an additional 12 h. The control rats received the same lipid emulsion as the experimental rats, but without L-81 added, for 8 h. Lymph lipid was determined both by radioactivity and by glyceride-glycerol determination, and the apoA-I and apoA-IV concentrations were determined by rocket electroimmunophoresis assay. L-81 inhibited the rise in lymphatic lipid and apoA-IV output in the experimental rats after the beginning of lipid + L-81 infusion. Upon cessation of L-81 infusion, the mucosal lipid accumulated as a result of L-81 treatment was rapidly cleared into lymph as CM. This was associated with a marked increase in apoA-IV output; the maximal output was about 3 times that of the fasting level. There was a time lag of 4-5 h between the peak lymph lipid output and the peak lymph apoA-IV output during the unblocking phase in the experimental rats. There was also a comparable time lag between the maximal lipid and apoA-IV outputs in the control animals. Incorporation studies using [3H]leucine showed that apoA-IV synthesis was not stimulated during lipid + L-81 infusion, perhaps explaining the lack of increase in lymphatic A-IV secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein

Atherosclerosis is a state of heightened oxidative stress. Oxidized LDL is present in atherosclerotic lesions and used as marker for coronary artery disease, although in human lesions lipids associated with HDL are as oxidized as those of LDL. Here we investigated specific changes occurring to apolipoprotein A-I (apoA-I) and apoA-II, as isolated HDL and human plasma undergo mild, chemically induced oxidation, or autoxidation. During such oxidation, Met residues in apoA-I and apoA-II become selectively and consecutively oxidized to their respective Met sulfoxide (MetO) forms that can be separated by HPLC. Placing plasma at -20 degrees C prevents autoxidation, whereas metal chelators and butylated hydroxytoluene offer partial protection. Independent of the oxidation conditions, apoA-I and apoA-II (dimer) with two MetO residues accumulate as relatively stable oxidation products. Compared to controls, serum samples from subjects with the endothelial cell nitric oxide synthase a/b genotype that is associated with increased coronary artery disease contain increased concentrations of apoA-I with two MetO residues. Our results show that during the early stages, oxidation of HDL gives rise to specifically oxidized forms of apoA-I and apoA-II, some of which may be useful markers of in vivo HDL oxidation, and hence potentially atherosclerosis.     

Effects of apolipoproteins A-IV and A-I on the uptake of phospholipid liposomes by hepatocytes

We examined the effects of apolipoproteins A-IV and A-I on the catabolism of whole particles by hepatoma G2 cells and cultured primary hepatocytes. For this type of experiment, high density lipoprotein is unsuitable, because all of its lipid and protein components independently dissociate and exchange and hence poorly trace whole particle catabolism. We therefore used phosphatidylcholine liposomes with radioactive tracers entrapped within their aqueous cores. Apolipoproteins A-IV, A-I, or E added to liposomes became liposome-associated and produced no detectable release of encapsulated label. As a positive control, apolipoprotein E doubled the uptake of labeled liposomes by hepatoma cells, compared to apolipoprotein-free controls, and this increase could be blocked by the addition of excess unlabeled low density lipoprotein. Degradation of labeled liposomes by hepatoma cells was increased 6-fold by the addition of apolipoprotein E. In contrast, neither apolipoprotein A-IV nor A-I increased cellular uptake or degradation of the particles. Similar results were obtained with primary hepatocytes. In studies using apolipoprotein combinations, apolipoproteins A-IV and A-I were each able to displace apolipoprotein E from liposomes and thereby reduce cellular uptake. Our data indicate that apolipoproteins A-IV and A-I do not facilitate uptake or degradation of whole particles by liver-derived cells in vitro. However, these apolipoproteins may modulate receptor-mediated uptake of particles by reducing the amount of particle-bound apolipoprotein E.     

Serum opacity factor, a streptococcal virulence factor that binds to apolipoproteins A-I and A-II and disrupts high density lipoprotein structure

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.     

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.     

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.