Human apolipoprotein A-I liberated from high-density lipoprotein without denaturation.

Apolipoprotein A-I (apoA-I) was liberated from human high-density lipoprotein (HDL) without exposure to organic solvents or chaotropic salts by the action of isolated insect hemolymph lipid transfer particle (LTP). LTP-catalyzed lipid redistribution results in transformation of HDL into larger, less dense particles accompanied by an overall decrease in HDL particle surface area:core volume ratio, giving rise to an excess of amphiphilic surface components. Preferential dissociation of apolipoprotein versus phospholipid and unesterified cholesterol from the particle surface results in apolipoprotein recovery in the bottom fraction following ultracentrifugation at a density = 1.23 g/mL. ApoA-I was then isolated from other contaminating HDL apolipoproteins by incubation with additional HDL in the absence of LTP, whereupon apolipoprotein A-II and the C apolipoproteins reassociate with the HDL surface by displacement of apoA-I. After a second density gradient ultracentrifugation, electrophoretically pure apoA-I was obtained. Sedimentation equilibrium experiments revealed that apoA-I isolated via this method exhibits a tendency to self-associate in an aqueous solution while its circular dichroism spectrum was indicative of a significant amount of alpha-helix. Both measurements are consistent with that observed on material prepared by denaturation/renaturation. The ability of apoA-I to activate lecithin:cholesterol acyltransferase was found to be similar to that of apoA-I isolated by conventional methods. The present results illustrate that LTP-mediated alteration in lipoprotein particle surface area leads to dissociation of substantial amounts of surface active apoprotein components, thus providing the opportunity to isolate apoA-I without the denaturation/renaturation steps common to all previous isolation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Predicting the structure of apolipoprotein A-I in reconstituted high-density lipoprotein disks.

In reconstituted high-density lipoproteins, apolipoprotein A-I and phosphatidylcholines combine to form disks in which the amphipathic alpha-helices of apolipoprotein A-1 bind to the edge of a lipid bilayer core, shielding the hydrophic lipid tails from the aqueous environment. We have employed experimental data, sequence analysis, and molecular modeling to construct an atomic model of such a reconstituted high-density lipoprotein disk consisting of two apolipoprotein A-I proteins and 160 palmitoyloleoylphosphatidylcholine lipids. The initial globular domain (1-47) of apolipoprotein A-I was excluded from the model, which was hydrated with an 8-A shell of water molecules. Molecular dynamics and simulated annealing were used to test the stability of the model. Both head-to-tail and head-to-head forms of a reconstituted high-density lipoprotein were simulated. In our simulations the protein contained and adhered to the lipid bilayer while providing good coverage of the lipid tails.     

Structure and function of apolipoprotein A-I and high-density lipoprotein

Structural biology and molecular modeling have provided intriguing insights into the atomic details of the lipid-associated structure of the major protein component of HDL, apo A-I. For the first time, an atomic resolution map is available for future studies of the molecular interactions of HDL in such biological processes as ABC1-regulated HDL assembly, LCAT activation, receptor binding, reverse lipid transport and HDL heterogeneity. Within the context of this paradigm, the current review summarizes the state of HDL research.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

The roles of C-terminal helices of human apolipoprotein A-I in formation of high-density lipoprotein particles

Apolipoprotein A-I (apoA-I) accepts cholesterol and phospholipids from ATP-binding cassette transporter A1 (ABCA1)-expressing cells to form high-density lipoprotein (HDL). Human apoA-I has two tertiary structural domains and the C-terminal domain (approximately amino acids 190–243) plays a key role in lipid binding. Although the high lipid affinity region of the C-terminal domain of apoA-I (residues 223–243) is essential for the HDL formation, the function of low lipid affinity region (residues 191–220) remains unclear. To evaluate the role of residues 191–220, we analyzed the structure, lipid binding properties, and HDL formation activity of Δ191–220 apoA-I, in comparison to wild-type and Δ223–243 apoA-I. Although deletion of residues 191–220 has a slight effect on the tertiary structure of apoA-I, the Δ191–220 variant showed intermediate behavior between wild-type and Δ223–243 regarding the formation of hydrophobic sites and lipid interaction through the C-terminal domain. Physicochemical analysis demonstrated that defective lipid binding of Δ191–220 apoA-I is due to the decreased ability to form α-helix structure which provides the energetic source for lipid binding. In addition, the ability to form HDL particles in vitro and induce cholesterol efflux from ABCA1-expressing cells of Δ191–220 apoA-I was also intermediate between wild-type and Δ223–243 apoA-I. These results suggest that despite possessing low lipid affinity, residues 191–220 play a role in enhancing the ability of apoA-I to bind to and solubilize lipids by forming α-helix upon lipid interaction. Our results demonstrate that the combination of low lipid affinity region and high lipid affinity region of apoA-I is required for efficient ABCA1-dependent HDL formation.     

Molecular packing of high-density and low-density lipoprotein surface lipids and apolipoprotein A-I binding

The surface pressure (pi)-molecular area (A) isotherms for monolayers of human high-density lipoprotein (HDL3) and low-density lipoprotein (LDL) phospholipids and of mixed monolayers of these phospholipids with cholesterol spread at the air-water interface were used to deduce the likely molecular packing at the surfaces of HDL3 and LDL particles. LDL phospholipids form more condensed monolayers than HDL3 phospholipids; for example, the molecular areas of LDL and HDL3 phospholipids at pi = 10 dyn/cm are 88 and 75 A2/molecule, respectively. The closer packing in the LDL phospholipids monolayer can be attributed to the higher contents of saturated phosphatidylcholines and sphingomyelin relative to HDL3. Cholesterol condenses both HDL3 and LDL phospholipid monolayers but has a greater condensing effect on the LDL phospholipid monolayer. The pi-A isotherms for mixed monolayer of HDL3 phospholipid/cholesterol and LDL phospholipid/cholesterol at stoichiometries similar to those at the surfaces of lipoprotein particles suggest that the monolayer at the surface of the LDL particle is significantly more condensed than that at the surface of the HDL3 particle. The closer lateral packing in LDL is due to at least three factors: (1) the difference in phospholipid composition; (2) the higher unesterified cholesterol content in LDL; and (3) a stronger interaction between cholesterol and LDL phospholipids relative to HDL3 phospholipids. The influence of lipid molecular packing on the affinity of human apolipoprotein A-I (apo A-I) for HDL3 and LDL surface lipids was evaluated by monitoring the adsorption of 14C-methylated apo A-I to monolayers of these lipids spread at various initial surface pressures (pi i).(ABSTRACT TRUNCATED AT 250 WORDS)     

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis

HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.     

Triiodothyronine increases rat apolipoprotein A-I synthesis and alters high-density lipoprotein composition in vivo

The effects of altered serum 3,3',5-triiodothyronine levels on rat lipoprotein metabolism were examined. Daily injections of the hormone (50 micrograms/100 g body mass) over a period of six days led to an increase of 6.4-fold in the hepatic mRNA level for apolipoprotein(apo)A-I, and a 21% increase in serum apoA-I levels. 12h after a single injection of 3,3',5-triiodothyronine the rate of [14C]leucine incorporation into apoA-I increased 2.1 fold. Conversely, in hypothyroid rats there was a decrease in hepatic mRNA levels for apoA-I and a decreased rate of [14C]leucine incorporation into apoA-I. The increase in hepatic apoA-I mRNA levels following 3,3',5-triiodothyronine treatment occurred prior to significant changes in serum triacylglycerol levels. High-density lipoprotein (HDL) particles isolated from the serum of hyperthyroid rats were smaller and enriched in apoA-I compared to apoA-IV and apoE. Similar changes in HDL composition were observed following in vitro incubations of normal rat serum with purified rat apoA-I. The results suggest that during altered thyroid status, changes in serum HDL size and composition occur in association with significant changes in apoA-I gene expression.     

Exclusive association of paraoxonase 1 with high-density lipoprotein particles in apolipoprotein A-I deficiency.

Paraoxonase1 (PON1) is a high-density lipoprotein (HDL)-associated protein which removes peroxidized lipids from lipoproteins. It has been proposed that apolipoprotein A-I (apoA-I) is an important determinant for its stabilization on HDL. However, little is known about its existence and activity in an apoA-I-deficient state in humans. To characterize the nature of PON1 in apoA-I deficiency, we investigated PON1 in an apoA-I-deficient patient. When serum was analyzed on fast protein liquid chromatography, PON1 protein was distributed almost exclusively on HDL despite the absence of apoA-I; on the other hand, 38.5% of PON1 protein was found in the lipoprotein-free fraction when the lipoproteins were fractionated through ultracentrifugation. The stability of PON1 activity in the patient serum was almost the same as in the normal control sera throughout incubation at 14 degrees C for 7 days. However, when the sera were incubated at 37 degrees C for 24 h, its activity declined more than those in the normal controls (19% versus 4% reduction of the initial values). Our results demonstrated that PON1 protein possesses a preferential association with HDL even in the absence of apoA-I, although apoA-I is a crucial factor for the maximal activity and stabilization of PON1.     

Cholesterol efflux from macrophages mediated by high-density lipoprotein subfractions, which differ principally in apolipoprotein A-I and apolipoprotein A-II ratios

High-density lipoprotein (HDL) was fractionated by preparative isoelectric focussing into six distinct subpopulations. The major difference between the subfractions was in the molar ratio of apolipoprotein A-I to apolipoprotein A-II, ranging from 2.1 to 0.5. The least acidic particles had little apolipoprotein A-II, were larger and contained the most lipid. The efflux capacity of the HDL subfractions was tested with mouse peritoneal macrophages and a mouse macrophage cell line (P388D1), either fed with acetylated low-density lipoprotein or free cholesterol. All the HDL subfractions were equally able to efflux cholesterol. The efflux was concentration dependant and linear for the first 6 h. The HDL subfractions bound with high affinity (Kd = 6.7-7.9 micrograms/ml) at 4 degrees C to the cell surface of P388D1 cells (211,000-359,000 sites/cell). Ligand blotting showed that all the HDL subfractions bound to membrane polypeptides at 60, 100, and 210 kDa. These HDL binding proteins may represent HDL receptors. In summary HDL particles, which differed principally in ratio of apolipoprotein A-I to apolipoprotein A-II behaved in a similar manner for both cholesterol efflux and cell surface binding.     

Comparative models for human apolipoprotein A-I bound to lipid in discoidal high-density lipoprotein particles

We have constructed a series of models for apolipoprotein A-I (apo A-I) bound to discoidal high-density lipoprotein (HDL) particles, based upon the molecular belt model [Segrest, J. P., et al. (1999) J. Biol. Chem. 274, 31755-31758] and helical hairpin models [Rogers, D. P., et al. (1998) Biochemistry 37, 11714-11725], and compared these with picket fence models [Phillips, J. C., et al. (1997) Biophys. J. 73, 2337-2346]. Molecular belt models for discoidal HDL particles with differing diameters are presented, illustrating that the belt model can explain the discrete changes in HDL particle size observed experimentally. Hairpin models are discussed for the binding of apo A-I to discoidal HDL particles with diameters identical to those for the molecular belt model. Two models are presented for the binding of three monomers of apo A-I to a 150 A diameter discoidal HDL particle. In one model, two monomers of apo A-I bind to the exterior of the HDL particle in an antiparallel belt, with a third monomer of apo A-I bound to the disk in a hairpin conformation. In the second model, all three monomers of apo A-I are bound to the discoidal HDL particle in a hairpin conformation. Previously published experimental data for each model are reviewed, with FRET favoring either the belt or hairpin models over the picket fence models for HDL particles with diameters of 105 A. Naturally occurring mutations appear to favor the belt model for the 105 A particles, while the 150 A HDL particles favor the presence of at least one hairpin.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Raman spectroscopy of the thermal properties of reassembled high-density lipoprotein: apolipoprotein A-I complexes of dimyristoylphosphatidylcholine

Isolated complexes of apolipoprotein A-I (apoA-I), the major apoprotein of human plasma high-density lipoproteins, and dimyristoylphosphatidylcholine (DMPC) have been prepared and studied by differential scanning calorimetry (DSC) and Raman spectroscopy. DSC studies establish that complexes having lipid to protein ratios of 200, 100, and 50 to 1 each exhibit a broad reversible thermal transition at Tc = 27 degrees C. The enthalpy of lipid melting for each of the three complexes is about 3 kcal/mol of DMPC. Raman spectroscopy indicates that the physical state of lipid molecules in the complexes is different from that in DMPC multilamellar liposomes. Analysis of the C-H stretching region (2800-3000 cm-1) of the complexes and of the pure components in water suggests that below 24 degrees C (Tc for DMPC) there is considerably less lateral order among lipid acyl chains in the complexes than in DMPC liposomes. Above 24 degrees C, these types of interactions appear to contribute equally or slightly less to the complex structure than in pure DMPC. The temperature dependence of peaks in the C-C stretching region (1000-1180 cm-1) reveals a continuous increase in the number of lipid acyl chain C-C gauche isomers over a broad range with increasing temperature. Compared to liposomes, DMPC in the complexes has more acyl chain trans isomers at temperatures above 24 degrees C; at temperatures above ca. 30 degrees C, trans isomer content is about the same for complexes and liposomes. A large change was observed in a protein vibrational band at 1340 cm-1 for pure vs. complexed apoA-I, indicating that protein hydrocarbon side chains are immobilized by lipid binding. The Raman data indicate that the reduction in melting enthalpy for complexes DMPC (approximately 3 kcal/mol) compared to that for free DMPC (approximately 6 kcal/mol) is due to reduced van der Waals interactions in the low-temperature lipid phase.     

Speciation of human plasma high-density lipoprotein (HDL): HDL stability and apolipoprotein A-I partitioning

The distribution of apolipoprotein (apo) A-I between human high-density lipoproteins (HDL) and water is an important component of reverse cholesterol transport and the atheroprotective effects of HDL. Chaotropic perturbation (CP) with guanidinium chloride (Gdm-Cl) reveals HDL instability by inducing the unfolding and transfer of apo A-I but not apo A-II into the aqueous phase while forming larger apo A-I deficient HDL-like particles and small amounts of cholesteryl ester-rich microemulsions (CERMs). Our kinetic and hydrodynamic studies of the CP of HDL species separated according to size and density show that (1) CP mediated an increase in HDL size, which involves quasi-fusion of surface and core lipids, and release of lipid-free apo A-I (these processes correlate linearly), (2) >94% of the HDL lipids remain with an apo A-I deficient particle, (3) apo A-II remains associated with a very stable HDL-like particle even at high levels of Gdm-Cl, and (4) apo A-I unfolding and transfer from HDL to water vary among HDL subfractions with the larger and more buoyant species exhibiting greater stability. Our data indicate that apo A-I's on small HDL (HDL-S) are highly dynamic and, relative to apo A-I on the larger more mature HDL, partition more readily into the aqueous phase, where they initiate the formation of new HDL species. Our data suggest that the greater instability of HDL-S generates free apo A-I and an apo A-I deficient HDL-S that readily fuses with the more stable HDL-L. Thus, the presence of HDL-L drives the CP remodeling of HDL to an equilibrium with even larger HDL-L and more lipid-free apo A-I than with either HDL-L or HDL-S alone. Moreover, according to dilution studies of HDL in 3 M Gdm-Cl, CP of HDL fits a model of apo A-I partitioning between HDL phospholipids and water that is controlled by the principal of opposing forces. These findings suggest that the size and relative amount of HDL lipid determine the HDL stability and the fraction of apo A-I that partitions into the aqueous phase where it is destined for interaction with ABCA1 transporters, thereby initiating reverse cholesterol transport or, alternatively, renal clearance.     

Serum opacity factor unmasks human plasma high-density lipoprotein instability via selective delipidation and apolipoprotein A-I desorption

Human plasma high-density lipoproteins (HDL) are important vehicles in reverse cholesterol transport, the cardioprotective mechanism by which peripheral tissue-cholesterol is transported to the liver for disposal. HDL is the target of serum opacity factor (SOF), a substance produced by Streptococcus pyogenes that turns mammalian serum cloudy. Using a recombinant (r) SOF, we studied opacification and its mechanism. rSOF catalyzes the partial disproportionation of HDL into a cholesteryl ester-rich microemulsion (CERM) and a new HDL-like particle, neo HDL, with the concomitant release of lipid-free (LF)-apo A-I. Opacification is unique; rSOF transfers apo E and nearly all neutral lipids of approximately 100,000 HDL particles into a single large CERM whose size increases with HDL-CE content (r approximately 100-250 nm) leaving a neo HDL that is enriched in PL (41%) and protein (48%), especially apo A-II. rSOF is potent; within 30 min at 37 degrees C, 10 nM rSOF opacifies 4 microM HDL. At respective low and high physiological HDL concentrations, LF-apo A-I is monomeric and tetrameric. CERM formation and apo A-I release have similar kinetics suggesting parallel or rapid sequential steps. According to the reaction products and kinetics, rSOF is a heterodivalent fusogenic protein that uses a docking site to displace apo A-I and bind to exposed CE surfaces on HDL; the resulting rSOF-HDL complex recruits additional HDL with its binding-delipidation site and through multiple fusion steps forms a CERM. rSOF may be a clinically useful and novel modality for improving reverse cholesterol transport. With apo E and a high CE content, CERM could transfer large amounts of cholesterol to the liver for disposal via the LDL receptor; neo HDL is likely a better acceptor of cellular cholesterol than HDL; LF-apo A-I could enhance efflux via the ATP-binding casette transporter ABCA1.     

Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.     

Interaction of human plasma high-density lipoprotein HDL2b with discoidal complexes of dimyristoylphosphatidylcholine and apolipoprotein A-I

The interaction of HDL2b, a major subclass (d = 1.063 - 1.100 g/ml) of human plasma high-density lipoproteins, with discoidal complexes composed of dimyristoylphosphatidylcholine (DMPC) and apolipoprotein A-I (weight ratio, DMPC/apolipoprotein A-I (2.1 - 2.5:1); dimensions, 10.0 x 4.4 nm) was investigated. Incubation at 37 degrees C for 4.5 h of HDL2b with discoidal complexes resulted in a transfer of DMPC from the discoidal complexes to the HDL2b, a release of lipid-free apolipoprotein A-I from the discoidal complexes during such transfer, and a dissociation of some apolipoprotein A-I from the HDL2b surface. The number of discoidal complexes degraded during interaction with HDL2b depended on the initial molar ratio of HDL2b to discoidal complexes. Approximately one molecule of HDL2b was required for the degradation of one discoidal complex particle, and the degradation process appeared limited by the capacity of the HDL2b for uptake of DMPC. Degradation of discoidal complexes was also observed when human plasma LDL (d = 1.006-1.063 g/ml) was substituted for HDL2b in the interaction mixture.     

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.     

Evidence in vitro that hepatic lipase reduces the concentration of apolipoprotein A-I in rabbit high-density lipoproteins

Incubation of rabbit plasma in vitro with hepatic lipase resulted in the hydrolysis of triacylglycerol in high-density lipoproteins (HDL) and a reduction in HDL particle size. These changes were accompanied by a decrease in the concentration of apolipoprotein A-I (apo A-I) in the HDL. The loss of apo A-I was demonstrated independently by ultracentrifugation, size exclusion chromatography and gradient gel-immunoblot analysis. It was unrelated to hydrolysis of HDL phospholipids but did correlate with the reduction in HDL particle size. These studies suggest that the concentration of apo A-I in HDL may be influenced by factors which regulate the metabolism of HDL core lipid constituents.