Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.     

Rapid method for the isolation of two purified subfractions of high density lipoproteins by differential dextran sulfate-magnesium chloride precipitation

We describe a rapid and reliable three-step precipitation procedure for the isolation of large amounts of the two major components of high density lipoproteins (HDL) in human serum. Precipitation was accomplished by means of dextran sulfate (DS) of mol. wt. 500,000 and MgCl2. First, all apoB-associated lipoproteins of any density were selectively precipitated with critical concentrations of reagents. Secondly, a subfraction of HDL was differentially precipitated from the apoB-depleted serum by increasing the concentration of both reagents. Eventually, the bulk of the remainder of HDL was precipitated by lowering the pH to 5.4. According to the precipitation patterns and the density profiles, the DS-Mg procedure provides a clear differentiation between the two HDL components. According to the compositional criteria and the ultracentrifugal characteristics, the two polyanion-precipitated subclasses are very similar to, if not identical with, the two density subclasses, the lighter HDL2 and the heavier HDL3, isolated by preparative ultracentrifugation after apoB-containing lipoproteins had been removed.     

Spin labelling study of frozen solutions of porcine high density and low density lipoproteins: temperature dependence of the lipid dynamics

Frozen solutions of porcine lipoproteins, spin-labelled with steric acids, were studied as a function of temperature. High and low density lipoproteins and their subclasses were examined to obtain informatin on the relationship between the size, composition and dynamics of their lipid constituents. in low density lipoproteins (LDL) the temperature at which spin-labelled fatty acids responded to increased temperature depended on the position of the nitroxide moiety on the fatty acid chain. In LDL, e.s.r. spectra of 16-doxyl stearic acid I(1/14) with the nitroxide moiety buried depp in the phospholipid interior, responded to moderate increase of temperature even at ?50°C. In high densitylipoproteins (HDL), all spin-labelled fatty acids, I(m/n), remained in the frozen state (on the e.s.r. time scale) up to the melting point of the baffer. These differences in behaviour of the frozen lipoprotein solutions indicated that physicochemical properties of the surface constituents of the lipoprotein particle might be of a different nature in HLD and LDL.     

Formation of cholesterol- and apoprotein E-enriched high density lipoproteins in vitro

The delivery of cholesterol to canine serum or plasma altered the distribution of cholesterol and apoproteins in subclasses of high density lipoproteins (HDL). In these experiments, two in vitro systems were employed. The first system used cholesterol-celite particles to deliver cholesterol to canine plasma during 4-h incubations. When the cholesterol distribution in the lipoproteins was analyzed by Geon-Pevikon electrophoresis, an increase in cholesterol content was found in the slower migrating subclasses of HDL (HDL1 and HDLc). A large increase in apoprotein E (apo-E) was also observed in the lipoproteins. Densitometric analysis of lipid-stained, 4 to 30% gradient acrylamide gels of canine plasma after incubation with cholesterol-celite revealed that the concentration of the major high density lipoproteins (HDL3) decreased, and the concentration of subclasses of HDL-with apo-E (HDL1 and HDLc) increased 2- to 5-fold. In the second system, cholesterol-loaded mouse peritoneal macrophages released cholesterol to HDL in an incubation medium containing 10 to 20% canine serum. The HDL1 and HDLc, which demonstrated slower electrophoretic mobility as determined by Geon-Pevikon block electrophoresis, became enriched in cholesterol and cholesteryl esters. Gradient gel electrophoresis showed substantial increases in these subclasses of HDL-with apo-E. The cholesterol-loaded mouse peritoneal macrophages synthesized and secreted apo-E into the medium. When L-[35S]methionine was used as a precursor, 65 to 90% of the 35S-labeled protein associated with the lipoproteins in the 1.02 to 1.09 density range was immunoprecipitated with antibody directed against rat apo-E. Gradient gel electrophoresis of density fractions demonstrated the presence of HDL1 and HDLc as the major lipoproteins. In addition, when canine 125I-HDL3 (primarily apo-A-I-containing HDL) were added to canine serum and incubated with cholesterol-loaded macrophages, the appearance of HDL1 and HDLc was associated with a marked increase in the 125I label in these newly formed, cholesteryl ester-rich lipoproteins. There was a corresponding marked reduction in the 125I-HDL3 in the serum. Similar results were observed using human HDL3 and human serum.     

Electrophoretic separation of high density lipoprotein subclasses in the presence of sodium tetraphenylborate]

It has been shown that the synthetic lipophilic anion, sodium tetraphenylboron, used at concentrations up to 15 mM causes modification of high density lipoproteins (HDL) by changing the electrophoretic properties of individual lipoprotein subclasses. The densitograms obtained after separation of sodium tetraphenylborate-modified HDL under non-denaturing conditions have a better resolution with respect to subclasses 2a and 2b, which markedly facilitates the analysis of the subfractional composition of lipoproteins.     

Interchange of apoprotein components between the human plasma high density lipoprotein subclasses HDL2 and HDL3 in vitro.

The major apoproteins of human high density lipoproteins (HDL) labeled with 125I have been shown to exchange between the two major HDL subclasses HDL2 and HDL3 in vitro. This bidirectional exchange process is inhibited by cross-linking with bifunctional reagents and is apparently dependent upon the formation of collision complexes. This exchange has been demonstrated both when the subclasses of HDL are free in solution and also when one of them is covalently bound to Sepharose. Using system involving Sepharose-bound HDL, it could be shown that not only free apoprotein molecules but subunits consisting of lipid-apoprotein combinations were exchanged between HDL2 and HDL3. The rate of exchange in these processes is significant in the lifetime of the protein particles in vivo equalling approximately 2.5% per h for apoprotein exchange. These experiments suggest that there is a dynamic relationship between HDL2 and HDL3 even though each of them exists alone in vitro as stable separate entities; when they are placed together in solution significant interaction occurs between the particles. Apoprotein exchange occurs between HDL2:HDL2 and HDL3:HDL3 as well as between HDL2 and HDL3 molecules. These data also suggest that the interconversion of HDL2 and HDL3 may be affected by the availability of lipids.     

Binding characteristics of high density lipoprotein subclasses to porcine liver, adrenal and skeletal muscle plasma membranes

1. We compared binding characteristics of 125I-labeled high density lipoprotein (HDL) subclasses to porcine liver, adrenal and skeletal muscle plasma membranes. 2. HDL subclasses were discriminated by their buoyant densities (HDL2 and HDL3) or by their apolipoprotein (apo) content (Lp-AI (particles containing apoA-I but no apoA-II) and LpA-I/A-II (particles containing both apoA-I and apoA-II)). 3. HDL2 and HDL3 showed saturable binding to the three types of membrane preparations. 4. No differences were found in the Kds within one HDL subclass. 5. Kds and maximal binding of HDL2 were lower than these of HDL3. Unlabeled HDL2 and HDL3, but not LDL, effectively displaced 125I-HDL2 and 125I-HDL3. 6. Binding of HDL was independent of the concentration of NaCl and did not require calcium. 7. These results suggest a process mediated by a single specific receptor in porcine liver, adrenal and skeletal muscle plasma membranes. 8. We also studied binding characteristics of HDL subclasses Lp-AI and LpA-I/A-II to porcine liver membranes. LpA-I showed the highest Kd and maximal binding. 9. All types of HDL subclasses studied (i.e. HDL2, HDL3, LpA-I and LpA-I/A-II) effectively competed for binding of both Lp-AI and LpA-I/A-II, suggesting that the HDL subclasses studied bind to the same receptor by their apoA-I moiety.     

Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation

The major lipoprotein density classes (chylomicrons-VLDL, LDL, HDL(2) and HDL(3)) were isolated from human plasma in a two-step ultracentrifugal procedure using the Ti-14 zonal rotor. The isolation of the two major high density lipoprotein subclasses (HDL(2) and HDL(3)) was achieved in a 24-hr run using a nonlinear NaBr gradient in the density range of 1.00-1.40. The lipoproteins with a density < 1.063 found in the rotor's center were isolated in a second run of 140 min duration using a continuous linear NaBr gradient in the density range of 1.00-1.30. The isolated lipoproteins were analyzed for chemical composition and for electrophoretic mobility; purity of isolated fractions was checked by immunochemistry. The lipoproteins exhibited flotation rates, chemical compositions, and molecular weights similar to those found with the common sequential procedures in angle-head rotors. The amount of lipoprotein lipids in the bottom fraction of the zonal rotor was comparable to that of the angle-head rotor. The described method yields the main lipoprotein density classes free from albumin in a very short running time; compared with the rate-zonal techniques already in use, this method allows the quantitative separation of an additional lipoprotein density class (HDL(2)) without increasing the running time. Furthermore, this procedure proved to be suitable for isolation of plasma lipoproteins from subjects with various types and varying degrees of hyperlipoproteinemia.     

Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy

Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter time constant was attributed to motion of the chromophore within the lipoprotein particle, while the longer time constant represented the global tumbling of the particles in solution. The measured correlation times for this global motion were about twice those predicted from the Stokes-Einstein relationship. Covalent labeling of the apolipoproteins of the low and high density lipoproteins with erythrosin revealed the existence of segmental motion of labeled domains of the apolipoprotein within their respective particles. The correlation times for this motion were within the range 10-50 microseconds. The binding of low density lipoproteins to receptors on membranes isolated from the adrenal cortex resulted in a freezing of the global motion, but maintenance of the faster segmental motion of the labeled domains of the apolipoprotein. The experiments imply that in these membranes there is no global motion of the low density lipoprotein-receptor complex on the phosphorescence time scale. Similar results were found for the binding of high density lipoproteins to liver plasma membranes. The contributions of nonspecific binding of the labeled lipoproteins to the measured phosphorescence anisotropy were carefully assessed.     

Kinetics of tryptophan oxidation in plasma lipoproteins by myeloperoxidase-generated HOCl.

The relative susceptibility of the apoprotein components of human lipoproteins [high-density lipoprotein (HDL) and low-density lipoprotein (LDL)] and their subclasses to oxidation by the myeloperoxidase/H2O2/Cl- system in vitro was studied by measuring the decrease in rate of tryptophan fluorescence. Whereas the lipoprotein-modification rate showed a saturation type of dependence on the concentration of myeloperoxidase, a biphasic dependence on the concentration of the lipoproteins was found. High concentrations of H2O2 were also found to inhibit tryptophan oxidation in LDL but to a lesser extent in HDL. The optimal rate of LDL and HDL modification was observed at pH 6.0. HDL was modified much more rapidly than LDL, which may be due to differences in size and different relative contents of protein and lipids per particle. No differences in rates of modification of LDL subclasses were observed, when the assays were standardized to equal LDL protein concentrations, but, when standardized to equal particle mass, an optimum at subclass 8 was found, which is probably due to differences in apolipoprotein B-100 conformation. It was concluded that HDL may have a beneficial effect in retarding LDL modification in inflammatory processes.     

The binding of low density lipoprotein by liver membranes in the pig.

Porcine liver membranes are capable of high affinity binding of homologous low density lipoproteins (LDL). Binding is time and temperature dependant and substrate saturable. High affinity binding sites are half saturated at 11 μg/ml lipoprotein-protein. The binding of ¹²⁵I-LDL is inhibited by unlabelled homologous LDL, very low density lipoproteins (VLDL) and high density lipoproteins (HDL) and also be human LDL and HDL, but not by unrelated proteins tested. The binding and displacement patterns with membranes from several other porcine tissues are similar to those of liver membranes. These results suggest the presence of “lipoprotein binding sites” in liver membranes which recognize structural features common to the lipoproteins and further indicate that liver membranes are not unique in their ability to bind LDL.     

A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins

A new apolipoprotein, termed apolipoprotein J (apoJ), was purified from human plasma by immunoaffinity chromatography. ApoJ is a glycoprotein consisting of disulfide-linked subunits of 34-36 and 36-39 kDa. Each subunit is glycosylated and has a pI range of 4.9-5.4. ApoJ exists in the plasma associated with high density lipoproteins (HDL) and specifically with subclasses of HDL which also contain apoAI and cholesteryl ester transfer protein activity. Immunoaffinity purified apoJ-HDL subclasses have apparent molecular masses of 80, 160, 240, 340, and 520 kDa, as determined by gradient gel electrophoresis. By negative staining electron microscopy, apoJ-HDL range in diameter from 5 to 16 nm. Fractionation of plasma by vertical gradient density centrifugation revealed apoJ-HDL in HDL2 (d 1.063-1.125 g/ml) with the majority overlapping HDL3 (d 1.125-1.21 g/ml) and very high density lipoprotein (d 1.21-1.25 g/ml). The bimodal density distribution of apoJ-HDL suggests that these subclasses have a unique metabolic relationship and may play a role in the transport of cholesterol from peripheral tissues to the liver.     

Effect of fish oils on rat plasma lipoproteins

Plasma high (HDL) and low (LDL) density lipoproteins were isolated from rats fed a diet supplemented with either fish (menhaden) oil or hydrogenated coconut oil (control). Fluorescence polarization and electron spin resonance of labelled fatty acid probe molecules incorporated into the outer amphiphilic monolayer of HDL indicate molecular motion is restricted in the upper portion of the acyl chain following the fish oil diet, which is consistent with a 'hook' conformation predicted by preliminary molecular model calculations for n-3 fatty acids (the predominant component of fish oil). Negligible dependence on diet was observed in LDL. Thus, a HDL specific effect of dietary fish oil on molecular fluidity and order in the outer monolayer of rat lipoproteins is suggested.     

Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.     

Mechanism of the cholesteryl ester transfer protein-mediated uptake of high density lipoprotein cholesteryl esters by Hep G2 cells

Plasma cholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl esters (CE) between lipoproteins and was reported to also directly mediate the uptake of high density lipoprotein (HDL) CE by human Hep G2 cells and fibroblasts. The present study investigates that uptake and its relationship to a pathway for "selective uptake" of HDL CE that does not require CETP. HDL3 labeled in both the CE and apoprotein moieties was incubated with Hep G2 cells. During 4-h incubations, CE tracer was selectively taken up from doubly labeled HDL3 in excess of apoA-I tracer, and added CETP did not modify that uptake. However, during 18-20-h incubations, CETP stimulated the uptake of CE tracer more than 4-fold without modifying the uptake of apoA-I tracer. This suggested that secreted products, perhaps lipoproteins, might be required for the CETP effect. Four inhibitors of lipoprotein uptake via low density lipoprotein (LDL) receptors (heparin, monensin, an antibody against the LDL receptor, and antibodies against the receptor binding domains of apoB and apoE) effectively blocked the CETP stimulation of CE tracer uptake. Heparin caused an increase in CE tracer in a d less than 1.063 g/ml fraction of the medium that more than accounted for the heparin blockade of CETP-stimulated CE uptake. CETP did not affect the uptake of doubly labeled HDL3 by human fibroblasts, even at twice plasma levels of activity, and heparin did not modify uptake of HDL3 tracers. Thus the CETP effect on Hep G2 cells can be accounted for by transfer of HDL CE to secreted lipoproteins which are then retaken up, and there is no evidence for a direct effect of CETP on cellular uptake of HDL CE.     

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.     

The intracellular distribution of labelled lipoproteins in the tissues of rats under physical loading]

A study was made of the influence of a 3 hour physical loading on the intracellular distribution of 125I-labeled very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) in the rat tissues after intravenous injection. The uptake of VLDL by liver was decreased, that of LDL was not altered and that uptake of HDL was increased, Redistribution of labeled lipoproteins between the subcellular structures was found. Accumulation of labeled VLDL in cytosol, microsome and large membranes was observed in muscles. The radioactivity level of VLDL was decreased in lysosomes, whereas the level of HDL was increased, Physical loading increased the uptake of HDL in adrenal glands. Accumulation of radioactivity was found in cytosol and small membranes. The data confirm the important role of lipoproteins of cellular metabolism regulation.     

Differential Stability of High-density Lipoprotein Subclasses: Effects of Particle Size and Protein Composition

High-density lipoproteins (HDLs) are complexes of proteins (mainly apoA-I and apoA-II) and lipids that remove cholesterol and prevent atherosclerosis. Understanding the distinct properties of the heterogeneous HDL population may aid the development of new diagnostic tools and therapies for atherosclerosis. Mature human HDLs form two major subclasses differing in particle diameter and metabolic properties, HDL₂ (large) and HDL₃ (small). These subclasses are comprised of HDL(A-I) containing only apoA-I, and HDL(A-I/A-II) containing apoA-I and apoA-II. ApoA-I is strongly cardioprotective, but the function of the smaller, more hydrophobic apoA-II is unclear. ApoA-II is thought to counteract the cardioprotective action of apoA-I by stabilizing HDL particles and inhibiting their remodeling. To test this notion, we performed the first kinetic stability study of human HDL subclasses. The results revealed that the stability of plasma spherical HDL decreases with increasing particle diameter; which may facilitate preferential cholesterol ester uptake from large lipid-loaded HDL₂. Surprisingly, size-matched plasma HDL(A-I/A-II) showed comparable or slightly lower stability than HDL(A-I); this is consistent with the destabilization of model discoidal HDL observed upon increasing the A-II to A-I ratio. These results clarify the roles of the particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of apoA-II in this remodeling.     

Lipid metabolism in cultured cells. XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic and tumor virus-transformed human fibroblasts.

Serum lipoproteins control cell cholesterol content by regulating its uptake, biosynthesis, and excretion. Monolayers of cultured fibroblasts were used to study interactions with human high density (HDL) and low density (LDL) lipoproteins doubly labeled with [(3)H]cholesterol and (125)I in the apoprotein moiety. In the binding assay for LDL, the absence of specific LDL receptors in type II hypercholesterolemic fibroblasts was confirmed, whereas monolayers of virus-transformed human lung fibroblasts (VA-4) exhibited LDL binding characteristics essentially the same as normal lung fibroblasts. In the studies of HDL binding, specific HDL binding sites were demonstrated in normal and virus-transformed fibroblasts. In addition, type II hypercholesterolemic cells, despite the loss of LDL receptors, retained normal HDL binding sites. No significant competition was displayed between the two lipoprotein classes for their respective binding sites over a 5-fold concentration range. In VA-4 cells, the amount of lipoprotein required to saturate half the receptor sites was 3.5 micro g/ml (9 x 10(-9) M) for LDL and 9.1 micro g/ml (9 x 10(-8) M) for HDL. Pronase treatment reduced LDL binding by more than half but had no effect on HDL binding. Chloroquine, a lysomal enzyme inhibitor, stimulated net LDL uptake 3.5-fold by increasing internalized LDL but had essentially no effect on HDL uptake. Further experiments were conducted using doubly labeled lipoproteins to characterize the interaction of LDL and HDL with cells. While the cholesterol and protein moieties of LDL were incorporated into cells at similar rates, the uptake of the cholesterol moiety of HDL was 5 to 10 times more rapid than that of the protein component. Furthermore, the apoprotein component of LDL is extensively degraded following exposure, whereas the apoprotein moiety of HDL retains its macromolecular chromatographic characteristics. These results indicate that HDL and LDL bind to cultured cells at separate sites and that further processing of the two lipoprotein classes appears to take place by fundamentally different mechanisms.-Wu, J-D., J. Butler, and J. M. Bailey. Lipid metabolism in cultured cells XVIII. Comparative uptake of low density and high density lipoproteins by normal, hypercholesterolemic, and tumor virus-transformed human fibroblasts.     

Secretion of apolipoproteins in very low density and high density lipoproteins by perfused rat liver

The incorporation of labeled amino acids into the peptides of very low density lipoproteins (VLDL) and high density lipoproteins (HDL) secreted by perfused rat liver was studied using a Ringer-albumin solution in the perfusate in place of serum to diminish exchange of peptides between VLDL and HDL. Among the lipoproteins, the greatest release of protein, greatest incorporation of amino acid, and highest specific activity were found in VLDL. After separation of the delipidated peptides by electrophoresis on polyacrylamide gel, the incorporation into VLDL peptides was found to be 5-10 times as great as into HDL peptides. There was virtually no incorporation into the peptides of low density lipoproteins (LDL). Approximately 25% of the radioactivity incorporated into perfusate VLDL failed to enter the 13% polyacrylamide gel. The remaining radioactivity was distributed primarily among three peptide bands; one, found in the upper portion of the gel, contained 45% of the total, most of the remainder being found in two rapidly migrating bands. These three peptides appear to approximate those of human apo-C in relative electrophoretic mobility. Most of the HDL peptide radioactivity entering the running gel was found in a band that migrates slightly faster than the main VLDL band. A portion of the radioactivity of this major HDL band did not enter the running gel unless beta-mercaptoethanol was present. Greater separation of these two bands by polyacrylamide gel electrophoresis for 24 hr confirmed that the major bands in VLDL and in HDL were different. The rapidly moving peptides of HDL were found to contain very little radioactivity. Determination of the intensity of staining of carrier-free perfusate VLDL and HDL peptides produced a pattern similar to the incorporation of labeled amino acids. It is concluded that the rapidly moving peptides, which may contain activators of lipoprotein lipase, are only secreted as part of the VLDL.