Dissociation of apolipoprotein AI from apoprotein-lipid complexes and from high-density lipoproteins. A fluorescence study.

The dissociation of the apoprotein AI (apoAI) from the apoAI-dimyristoylglycerophosphocholine complex and from human high-density lipoproteins (HDL), was induced by incubation with guanidinium hydrochloride at concentrations between 0 M and 7 M. The kinetics and extent of denaturation were followed by monitoring both the fluidity of the lipid phase by fluorescence polarization measurements and the protein conformation by measuring the tryptophanyl fluorescence emission. The association with lipids protects apoAI against denaturation both in HDL and in the apoAI-phospholipid complex. The results of the kinetic and end-point measurements suggest that the denaturing effect of guanidine hydrochloride on the apoAI-lipid complex and on HDL is a two-step process. It involves the dissociation of the apoAI-phospholipid bond, as evidenced by fluidity measurements: this effect is maximal between 3 M and 4 M guanidine hydrochloride. The conformational change of the apoAI protein into a randomly coiled structure with the tryptophanyl residues exposed to the solvent is maximal between 4 M and 6 M guanidine hydrochloride. HDL and the apoAI-phospholipid complex have a closely similar behaviour towards denaturation by guanidine hydrochloride indicating that the phospholipid-apoAI association in HDL is primarily responsible for the stability of the lipoprotein molecule.

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol.

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.

In vitro interaction of human HDL with human apolipoprotein A-II. Synthesis of apolipoprotein A-II-rich HDL.

The aim of this study was to define the specific affinity of human apolipoproteins A-I and A-II for HDL lipids and to investigate the possible transfer of apolipoproteins from the HDL molecule. For this purpose we incubated human HDL with increasing amounts of isolated apolipoprotein A-II. After incubation the reaction products were separated by gel chromatography and apolipoproteins A-I and A-II were quantified separately by immunonephelometry and HDL lipids by thin-layer chromatography. According to our results, apolipoprotein A-II progressively displaces apolipoprotein A-I to generate an HDL-like particle with identical lipid composition, hydrodynamic properties and lipid fluidity. These data indicate that apolipoprotein A-II is able to displace quantitatively apolipoprotein A-I from HDL in vitro, and that such a mechanism might contribute to the regulation of the HDL2 in equilibrium or formed from HDL3 distribution in plasma.

Displacement of apo A-I by A-II in lipid-apoprotein complexes and human HDL.

The aim of this study was to define the specific affinity of human apo A-I and apo A-II for HDL lipids and to investigate the possible transfer of apoproteins from the HDL molecule. For this purpose we incubated apo A-I -- lipid complexes prepared "in vitro", as well as human HDL with increasing amounts of isolated apo A-II. After incubation the reaction products were separated by gradient ultracentrifugation and gel chromatography. The apoproteins were quantitated separately by immunonephelometry and the apo A-I content was monitored by measuring the intrinsic tryptophan fluorescence. These results suggest that apo A-II has a higher affinity than apo A-I for the lecithin-cholesterol vesicle and that 2 mol apo A-II are able to displace 1 mol apo A-I from the apo A-I lipid complexes. Analogous results were obtained with HDL where two mol apo A-II substitute to 1 mol apo A-I to yield en apo A-II - rich HDL with identical lipid composition, hydrodynamic properties and fluidity. Such a mechanism might contribute to the regulation of the HDL2 in equilibrium with HDL3 distribution in plasma.

Reassembly of human apoproteins A-I and A-II with unilamellar phosphatidylcholine-cholesterol liposomes. Association kinetics and characterization of the complexes.

The kinetics of association between the human apoprotein A-I and apoprotein A-II and cholesterol dimyristoyl phosphatidylcholine (DMPC) vesicles are compared in this study and the lipid-apoprotein complexes are characterized. The association kinetics are followed by turbidity measurements monitoring the decrease of the vesicular size and by fluorescence polarization measurements monitoring the decrease in the mobility of the phospholipid acyl chains during complex formation. The influence of the incubation temperature and of the cholesterol/DMPC ratio has been studied by both techniques. Under all incubation conditions the apoprotein A-II associates more readily with cholesterol-DMPC vesicles than apoprotein A-I, as the kinetics are faster and the complex yield larger. With both apoproteins optimal complex formation takes place around the phospholipid transition temperature and around 10 mol% cholesterol. The apoprotein A-I/lipid association seems restricted to this narrow range for the temperature and the cholesterol/DMPC ratio, while the apoprotein A-II still associates with vesicles containing 20 mol% cholesterol and at temperatures up to 32 degrees C. The lipid-apoprotein complexes were isolated by gradient ultracentrifugation and by gel chromatography. According to these data the apoprotein A-II associates more readily than apoprotein A-I with cholesterol-DMPC vesicles to form protein-rich complexes, whilst the optimal apoprotein A-I-lipid association requires a more disordered lipid structure.

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 1. Calorimetric and potentiometric titration of the native apoA-I protein and of the apoA-I protein-dimyristoyl lecithin complex.

The ionization behaviour of native apoA-I protein is compare to that of its complex with synthetic dimyristoyl lecithin in studies using calorimetric, potentiometric and spectrophotometric titration. In the presence of phospholipids, 10 out of 21 lysines together with 22 acidic residues are masked in the complex. All tyrosines remain accessible to titration below pH 13. The apparent ionization enthalpy of the 11 lysine residues is not affected by the presence of phospholipids. These data are consistent with discrete binding sites located in the apoprotein helical segments as suggested by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. A tentative localisation of lysine, arginine, aspartic acid and glutamic acid residues directly involved in phospholipid binding is suggested, assuming that such helical regions are involved in apoprotein-phospholipid association.

Ionization behaviour of native apolipoproteins and of their complexes with lecithin. 2. Potentiometric titration of the native apo-A-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin.

A comparison of the ionization behaviour of the human apoA-II, apoC-I, apoC-III proteins and of their complexes with dimyristoyl lecithin is based on potentiometric titration of the basic and acidic residues and spectrophotometric titration of the phenolic groups. Experimental data suggest that a number of lysine, arginine, aspartic acid and glutamic acid residues are masked in the complexes. For each of these amino acids and in all three proteins the number of masked residues is consistent with the content of those regions predicted to be involved in lipid binding by the model of Segrest et al. [FEBS Lett. 38, 247-253 (1974)]. These data taken together with the results of calorimetric and titration experiments with the apoA-I protein reported in the accompanying article [Rosseneu et al. (1977) Eur. J. Biochem. 79, 251-257] strongly support the general nature of the proposed model and further suggest that ionic interactions have some role in the formation of the dimyristoyl lecithin/apolipoprotein complexes.