Characterization of the high-density lipoprotein and its major apoprotein from human, canine, bovine and chicken plasma.

The high-density lipoproteins (HDL) from canine, bovine, and chicken plasma have been shown to contain almost exclusively the apolipoprotein A-I, while human HDL contains a second major component, the apolipoprotein A-II. Chemical cross-linking demonstrated that dog and chicken HDL contain three apolipoprotein A-I molecules per particle, while bovine HDL contain approximately six apolipoprotein A-I molecules per particle. By this method, the amount of protein in human HDL2 (d = 1.063-1.12) was found to be approximately 120 000 g/mol, while for human HDL3 (d = 1.12-1.21) a value of approximately 90 000 g/mol was obtained, suggesting that the protein complement of HDL2 and HDL3 differ by only one apolipoprotein A-I chain per particle. Comparison of the apolipoprotein A-I from various animal species indicated that the canine and human apolipoprotein A-I proteins were the most similar by fluorescence, self-association properties, and immunoreactivity. Cross-linking of chicken and bovine apolipoprotein A-I yielded patterns distinctly different from that obtained with the human or canine counterpart. It is concluded that the quaternary structure of the various species of HDL is not directly correlated with the degree of self-association found for the protein constituents.

Effects of heparin-induced lipolytic activity on the structure of rat high-density lipoprotein.

Following its secretion into the plasma compartment, the high-density lipoprotein (HDL) is presumed to be acted upon by both soluble enzymes, such as lecithin:cholesterol acyltransferase (LCAT), and membrane-associated enzymes, such as lipoprotein lipase and hepatic lipase. Rats were injected intravenously with heparin to release membrane-associated lipolytic activities into the circulation and the collected plasma was incubated overnight at 37 degrees C in the presence or absence of an LCAT inhibitor or an inhibitor of lipoprotein lipase (1 M NaCl). It was observed that lipoprotein lipase accounted for most of the triglyceride hydrolase activity in the heparin-treated plasma, and that the heparin-releasable activities caused an increase in HDL density but no measurable change in particle size when LCAT was inhibited. Heparin treatment caused about a 60% decrease in plasma triacylglycerol during the interval between injection of heparin and blood collection. Although this caused marked compositional changes in the d less than 1.063 g/ml lipoproteins, no changes were observed in the lipid composition or apoprotein distribution in the HDL. Subsequent incubation for 18 h at 37 degrees C produced marked increases in the apoE content of HDL from heparin-treated plasma even when LCAT was inhibited. Time-course studies showed that in the presence of an LCAT inhibitor there was considerable conversion of phosphatidylcholine to lysophosphatidylcholine in heparin-treated plasma, and that this activity was diminished by 1 M NaCl, but that no phospholipolysis was observed in control plasma. By contrast, both heparin-treated and control plasma possessed substantial triglyceride hydrolase activity. The concurrent action of lipases and LCAT was observed to reduce the maximum level of cholesterol esterification which could be achieved in the absence of lipase activity. It is concluded that changes in HDL particle size are mainly attributable to LCAT, but that lipase activities, which are either free in rat plasma or releasable by heparin, play a role in restructuring the phospholipid moiety and altering the protein composition of the HDL, especially with respect to apoE, a potential ligand to cellular receptors.

Hybrid association between human apolipoproteins A-I and A-II in aqueous solution and in phospholipid recombinants.

The formation of hybrid association products between apolipoprotein A-I and apolipoprotein A-II from human high-density lipoprotein was investigated in solutions of these apolipoprotein and in recombinant particles with dimyristoylphosphatidylcholine (DMPC). It was found that these two proteins interact in solution to form hybrid association products, but not to a marked degree. When these two proteins were incubated together with DMPC, it was likewise found that there was little tendency to reside on the same particle, as judged from the absence of hybrid oligomers by chemical cross-linking. By a modified immunoelectrophoretic method it was found that only about 15% of the A-II and 10% of the A-I were precipitated by the heterologous antiserum; from this it is concluded that 80-90% of these proteins do not form hybrid recombinants with the other protein. These results suggest that in the delipidated state, as well as in discoidal recombinants, there do not exist strong protein-protein interactions between A-I and A-II. This implies that even in the high-density lipoprotein, where both proteins coexist in the same particle, the A-II does not stabilize the molecular structure through interactions with A-I, and its role in this molecule remains obscure.

Properties of phosphatidylethanolamine-containing phospholipid-apolipoprotein complexes modified by lecithin-cholesterol acyltransferase.

The effect of the inclusion of phosphatidylethanolamine (PE), a phospholipid with unusual packing properties, on the substrate properties of protein-lipid complexes toward lecithin-cholesterol acyltransferase (LCAT) has been studied. Recombinant particles of apolipoprotein A-I with dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and cholesterol were prepared at a molar ratio of 1:140:14 (A-I/DMPC/cholesterol) or 1:70:70:14 (A-I/DMPC/DLPE/cholesterol); the efficiency of cholesterol incorporation into complexes containing phosphatidylethanolamine was found to be very pH-dependent, with enhanced cholesterol incorporation at elevated pH values. By incubating the complexes with either purified human LCAT or the d greater than 1.21 g/ml fraction of rat serum as a source of LCAT activity, it was found that a high degree of cholesterol esterification could be achieved with either complex; however, the DLPE-containing complex possessed a much smaller Stokes' diameter than the DMPC-only particle despite compositional similarities between these complexes. With respect to particle diameter the DLPE-containing particles behaved more like complexes prepared with egg yolk lecithin than did complexes prepared with DMPC alone. When human LDL was added to the incubations to provide a source of additional cholesterol, the products were markedly different. Concomitant with an increased cholesteryl ester core was an increase in the protein stoichiometry in both types of particles, from 2 to 3 or 4 apo A-I per particle. The proportion of DLPE to DMPC in the products was reduced from 1:1 to 0.3:1, reflecting a preferential hydrolysis of PE by LCAT, and the Stokes' diameters of the DMPC-only and the DLPE-containing complexes were closely similar. We conclude that the presence of elevated proportions of certain phospholipid species may significantly alter both the physical properties of the particles and their substrate properties with regard to reactions with enzymes of lipid metabolism.

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.

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.

Proteolysis in vitro of low and high density lipoproteins in human plasma by Cerastes cerastes (Egyptian sand viper) venom.

Envenomation by snake venoms would be expected to result in proteolysis of plasma proteins as well as of cellular constituents. Incubation of human serum with crude venom from Cerastes cerastes showed that the plasma lipoproteins were a target of this venom. Fractionation of the crude venom by gel filtration revealed that high density lipoprotein (HDL) was susceptible almost exclusively to the highest mol. wt fraction of venom and that proteolysis was due to a metalloprotease. Although HDL was degraded only by this metalloprotease, the low density lipoprotein (LDL) was proteolyzed by both metalloproteases and serine proteases present in several fractions of the venom. Despite extensive degradation, LDL remained intact, as judged by gradient gel electrophoresis. The selectivity of venom fractions may prove useful in the study of lipoprotein structure.

Membrane cholesterol uptake by recombinant lipoproteins.

Recombinant lipoproteins, prepared with apo A-I isolated from human high density lipoprotein (HDL) and various phospholipids (PLs), were compared with respect to their ability to remove cholesterol (Chol) from labelled erythrocyte ghost membranes. It was found that uptake of Chol was essentially complete following an 8 h incubation at 37 degrees C. Quantitation of the amount of cholesterol taken up showed that recombinants prepared from bovine brain sphingomyelin (BBSM) or dipalmitoyl phosphatidylcholine (DPPC) acquired the highest proportion of Chol (80-140 mol/mol protein), whereas shorter chain phospholipids like dimyristoyl phosphatidylcholine (DMPC) acquired little or no membrane Chol. Chemical analysis of the incubation products indicated that this latter result was due to loss of PL, presumably to the membrane, with consequent disruption of the recombinant particle. Results with DPPC:A-I recombinants of differing PL/protein ratios and sizes showed that Chol uptake was fairly constant at 0.70 mol Chol/mol PL. It is concluded that discoidal, phospholipid-rich recombinant lipoproteins can effectively take up substantial amounts of Chol from physiological membranes, provided that the PLs utilized form micellar complexes which are capable of retaining their structural integrity during the incubation with the membranes.

Structural modifications of rat serum high density lipoprotein by pancreatic phospholipase A2.

An important and unusual aspect of the high density lipoprotein (HDL) in the rat is its tendency to undergo marked alterations in structure in response to physiological perturbations. In this study, the role of the surface lipids for maintenance of HDL integrity were investigated. Hydrolysis by pancreatic phospholipase A2 of the phospholipids of rat HDL in the presence of the d greater than 1.21 g/ml fraction of rat serum results in an increase in the particle diameter and an uptake of apo-E and apo A-IV from the lipoprotein-free fraction; augmentation of the albumin concentration in the incubation mixture intensified the observed changes, probably due to enhancement of the compositional changes brought about by phospholipase treatment. Phospholipase A2, treatment of the d less than 1.21 g/ml fraction of rat serum produces only minor changes in the properties of the isolated HDL. These data suggest that changes in apoprotein content reflect an uptake of A-IV and E by the rat HDL, rather than a net loss of apo A-I. Likewise, titration of the action of pancreatic phospholipase A2 on HDL apoprotein composition showed that initially a modest increase in apo A-IV content occurred, but with more extensive phospholipolysis there was a considerably greater increase in the apo-E content of the particle. The data suggest that hydrolysis of phospholipids such as occurs physiologically through the action of lecithin:cholesterol acyl transferase and hepatic lipase may alter the HDL structure independently from changes effected in the neutral lipid core.

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.

Selective proteolytic digestion as a method for the modification of human HDL3 structure.

Trypsin digestion of human high density lipoprotein (d 1.125-1.21 g/ml) on which the lysine residues have been masked with the reversible blocking group, 2,3,4,5-tetrahydrophthallic anhydride (THPA), was found to result in the fragmentation of the apoA-I component, but not the apoA-II component of this lipoprotein particle. Approximately 50-80% of the apoA-I polypeptide was found in a lipid-free fraction, while the residual apoA-I material plus the apoA-II moiety constituted a core particle that contained most of the original lipid. Immunological analysis indicated that such fragmentation did not affect the immunoreactivity of apoA-II, but that all immunoreactivity of apoA-I was lost within the first 30 min of trypsinization. By column chromatography and electron microscopy this core particle appeared identical in size with the untrypsinized THPA-modified HDL(3) material. Size analysis of the core particle peptides suggests that not all of the A-I molecules present on the HDL(3) are trypsinized to the same extent, which indicates possible nonequivalence of these peptide chains. Analysis of the amino acid composition revealed a somewhat greater proportion of hydrophobic residues in the lipid-bound fraction than in the lipid-free fraction. Analysis of tryptophan showed that almost all of this highly hydrophobic residue was found in the lipid-bound fraction; this suggests that lipid binding occurs preferentially in the more hydrophobic domains of the A-I molecule. Incubation of the core particle with intact apoA-I, obtained from either human or bovine HDL, showed that these proteins could be incorporated to regenerate an HDL(3) of selectively altered protein composition, compared to the original lipoprotein. It is concluded that some latitude is allowable in the surface/volume relationship in lipoproteins before reorganization of the particle is required; this might, for example, provide a mechanism whereby the HDL could serve in a storage role for the C apolipoproteins in plasma.-Swaney, J. B. Selective proteolytic digestion as a method for the modification of human HDL(3) structure.

Properties of lipid-apolipoprotein association products. Complexes of human apo AI and binary phospholipid mixtures.

The products resulting from the association of human apo A-I with binary phospholipid mixtures of dimyristoyl phosphatidylcholine (DMPC) and either dipalmitoyl (DPPC) or distearoyl (DSPC) phosphatidylcholine have been isolated and characterized. Effective lipid . protein complex formation was found to occur at the onset temperature for melting of the gel state, and equal incorporation of both lipid components of the binary mixture was observed. Two sizes of products were obtained, one containing 2 A-I molecules per complex and the other containing 3; the proportions of these two products depended upon the initial phospholipid/protein ratio employed. these two product species were found to be resolvable by density gradient centrifugation as well as gel filtration, reflecting substantial differences in composition as well as size. The ratio of DMPC to DPPC or DSPC was the same in the isolated complexes as in the initial mixture, suggesting that th protein does not associate preferentially with the fluid phase lipid, but with lipid domains in which the components are randomly distributed. Electron microscopy of recombinant particles containing a 2:1 ratio (w/w) of DSPC to DMPC revealed stacks of discs whose thickness was proportionately greater than for discs containing DMPC alone. Also of significance was the finding that recombinant discs containing 3 A-I molecules possessed a diameter approximately 1.5 times larger than recombinant discs containing 2 A-I molecules. These data are consistent with the model for the recombinant particles described by Tall et al. (Tall, A.R., Small, D.M., Deckelbau, R.J., and Shipley, G.G. (1977) J. Biol. Chem. 252; 4701-4711), in which the phospholipid is found as a circular bilayer, the thickness of which is dependent upon the length of the acyl chain, and around which the protein is distributed as an annulus.

Mechanisms of protein-lipid interaction. Association of apolipoproteins A-I and A-II with binary phospholipid mixtures.

Studies of the recombination of apo A-I and apo A-II the major protein components of human high density lipoprotein, with binary mixtures of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidyl-choline (DSPC) were performed. Recombination was observed to occur only near the lower bound temperature of the phase transition for each mixture. Similar experiments using binary mixtures of DMPC and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) demonstrated that recombination occurs in a temperature range which is believed to approximate the lower bound of the phase transition for this mixture as well. The reactivity of both types of mixtures toward recombination with apolipoproteins was found to decrease with decreasing proportions of DMPC, even though the effect of DMPC was to decrease the transition temperature in DMPC/POPC mixtures and to increase the transition temperature in DMPC/DSPC mixtures. A mechanism for insertion of apolipoproteins into lipid bilayers is proposed in which the protein gains entry to the interior of the bilayer through a defect resulting from equilibrium fluctuations of state at the onset temperature of acyl chain melting.

High density lipoprotein subpopulations from galactosamine-treated rats and their transformation by lecithin:cholesterol acyltransferase.

It is known that an acute hepatotoxicity is produced in rats by intraperitoneal administration of galactosamine; a consequence of this treatment is a marked deficiency of lecithin:cholesterol acyltransferase (LCAT) activity in the plasma compartment. In this study high density lipoprotein (HDL) from galactosamine-treated rats was isolated, resolved into subpopulations, and characterized. In contrast to HDL from control rats, which elutes from gel filtration columns as a single peak and has a diameter of 13.1 nm, HDL from the galactosamine-treated animals was found to elute in five major zones with diameters of 7.8-35 nm. Characterization of these subpopulations has revealed that the larger fractions are enriched in apolipoprotein E, phospholipid, and cholesterol, but contain little cholesteryl ester, while the smallest two fractions contain mainly apolipoprotein A-I, are enriched in phospholipid, and have 50-60% of their cholesterol in the ester form. Incubation of HDL from treated rats with a source of LCAT activity plus low and very low density lipoproteins caused transformation of these subpopulations into a species which, by size and composition, was essentially identical to control rat HDL. In addition, when the subpopulations were individually incubated with purified human lecithin:cholesterol acyltransferase and bovine serum albumin, there was a similar convergence toward a moderate particle size approximating control rat HDL. Cross-linking studies showed that incubation with LCAT activity reduced the heterogeneity of the treated rat HDL. We conclude that the galactosamine treatment induces a complex mixture of HDL that bears strong similarities to the small, apoA-I rich and large, apoE-rich particles seen in LCAT deficiency or secreted by hepatic cells in culture. Furthermore, these species appear to coalesce in the presence of the d greater than 1.21 g/ml fraction of control serum to yield a fairly homogeneous population that resembles control rat HDL in size, composition, and apoprotein content.

A rapid method for the synthesis of protein-lipid complexes using adsorption chromatography.

A novel and rapid method for the detergent-mediated synthesis of protein-lipid complexes has been developed and has several advantages over detergent dialysis methods. This new method involves co-incubation of human apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins (HDL), and dipalmitoylphosphatidylcholine for 1 hr in the presence of cholate, after which removal of greater than 99.7% of the detergent is achieved by a 2-hr batch adsorptive chromatography procedure. Complexes prepared by this method had a density of 1.10 g/ml, similar to plasma HDL. Chemical cross-linking of these products demonstrated that there was complete conversion of apoA-I to a protein-lipid complex that contained two molecules of apoA-I. One major band was resolved by gradient gel electrophoresis in the region of the gel expected for newly synthesized HDL. Results are described which show the application of this method to the study of lipid variation on the structure of model HDL, including the alteration of lipid-protein molar ratios and the addition of cholesterol.

Structural and functional domains of apolipoprotein A-I within high density lipoproteins.

We prepared discoidal and spherical model high density lipoprotein (HDL) with apolipoprotein A-I and 1-palmitoyl-2-oleoyl phosphatidylcholine at various lipid:protein ratios and compared their reactivity with exo- and endopeptidases to that of human HDL2 and HDL3. Limited proteolysis with trypsin, Staphylococcus V8 protease, and elastase yielded a major stable peptide of 11,000-11,500 daltons under conditions which completely degraded lipid-free A-I. By Western blotting this protease-resistant fragment was shown to consist of the amino-terminal 90-100 residues of the A-I protein; the residues on the carboxyl side of this peptide are therefore protease-sensitive and appear to correlate with the putative "hinge" region, which is especially reactive with antibodies. The amino terminus was very resistant to digestion with a variety of aminopeptidases, whereas carboxypeptidases could remove up to 70 residues from the lipid-free A-I protein or 12-24 residues from A-I in various HDL. When these truncated forms of A-I, in combination with lipid, were used to examine binding interactions with rat hepatic plasma membranes, it was found that removal of up to 20-24 residues from the carboxyl terminus had no significant effect on binding, whereas removal of 70 residues completely eliminated specific binding to the membranes. Taken together, our data indicate that there is a protease-resistant domain constituted by the first 90 residues of A-I, which, in HDL, contain little of the class of amphipathic helix characteristic of the rest of the molecule and most likely form a structure dominated by protein-protein interactions. At the carboxyl end of the protein, there is a functional domain constituted by residues 149-219 that possesses the capacity to bind to proteins on hepatic membranes.

Thermal dependence of apolipoprotein A-I-phospholipid recombination.

Studies of the recombination of apolipoprotein A-I (apo A-I), the major protein constituent of human high-density lipoprotein, and various synthetic phospholipids, both alone and in mixtures, have been performed. Pure diacyl phospholipids containing homologous fatty acids of the C12, C13, C14, and C15 series, as well as the two positional isomers containing C14 and C16 fatty acids in positions 1 and 2, undergo reaction with the apo A-I protein only near their gel-liquid-crystalline transition temperatures; the degree of reactivity of these phospholipids toward recombination was observed to decrease as the transition temperature increased. The presence of lysolecithin in the incubation mixtures at proportions of 5 mol/mol of protein or lower was not found to have a significant effect on the rate of recombination. Binary mixtures of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine at various proportions react maximally with apo A-I at the onset of the phase transition, as judged by comparison with published phase diagrams; in this case also, the rate of recombination was observed to decline for mixtures with higher phase transition temperatures. These results are interpreted in terms of protein insertion at lattice defects arising from the presence of phospholipid clusters undergoing the phase transition; these clusters are derived from the cooperative and simultaneous melting of a number of molecules, the cooperativity being dependent upon the nature of the phospholipid. It is postulated that phospholipids which melt in a more highly cooperative fashion are more capable of interacting with the apolipoproteins since these phospholipids will possess larger lattice defects during the phase transition.

Cross-linking studies of the self-association properties of apo-A-I and apo-A-II from human high density lipoprotein.

The state of self-association of the apoprotein components of human high density lipoprotein have been studied by use of the cross-linking reagent dimethyl-suberimidate. Analusis of the cross-linked products was carried out by soduim dodecyl sulfate-gel electrophoresis and by agarose column chromatography in 6 M guanidine hydrochloride. Apo-A-I was found to exist as a monomer at low concentration, but associates to tetrameric and pentameric forms at concentrations of 0.5 mg/ml or higher. The self-association was found to be ionic strength-dependent, with association promoted by the presence of salt. Apo-A-II was also found to associate, but the major oligomeric form observed was dimeric (Mr = 34,000), and the association was less dependent on ionic strength than for apo-A-I. Cross-linking in the presence of various concentrations of guanidine hydrochloride showed that apo-A-II self-association persisted at higher concentrations of the denaturant than for apo-A-I. Studies of the effect of temperature demonstrated that the self-association of both proteins was diminished at temperatures above 30 degrees C. Recombination of apo-A-II with phospholipid resulted in the formation of particles which yielded primarily trimers upon cross-linking. This suggests that phospholipid binding causes major reorganization of the self-associated forms of apo-A-II.