Replacement of Endogenous Cholesteryl Esters of Low Density Lipoprotein with Exogenous Cholesteryl Linoleate

Transcription

1 Replacement of Endogenous Cholesteryl Esters of Low Density Lipoprotein with Exogenous Cholesteryl Linoleate RECONSTITUTION OF A BIOLOGICALLY ACTIVE LIPOPROTEIN PARTICLE* (Received for publication, January 9, 1978) Monty Krieger,$ Michael S. Brown, Jerry R. Faust, and Joseph L. Goldstein From the Departments of Molecular Genetics and Internal Medicine, University of Texas Health Science Center at Dallas, Dallas, Texas The cholesteryl esters of human plasma low density lipoprotein (LDL) reside in a neutral lipid core surrounded by a polar shell consisting of phospholipids, unesterified cholesterol, and apoprotein B. In the current paper, we describe a procedure by which more than 99% of the core cholesteryl esters can be removed from the LDL particle by heptane extraction and replaced with an equal amount of exogenous cholesteryl linoleate. The reconstituted water-soluble LDL particle resembled native LDL with regard to the following physical and chemical properties: 1) similar composition with respect to relative proportions of total cholesterol, protein, and phospholipids; 2) p mobility on agarose gel electrophoresis; 3) susceptibility to precipitation by an antibody to native LDL; and 4) susceptibility to precipitation by heparin-manganese. The mean density of the reconstituted LDL (1.042 g/ml) was slightly greater than that of native LDL (1.034 g/ml) as determined by sucrose density gradient centrifugation. Studies of biological activity, as monitored in cultured human fibroblasts, showed that: 1) the reconstituted LDL was bound to the LDL receptor with the same affinity as that of native LDL, 2) the reconstituted LDL was taken up and hydrolyzed in lysosomes like native LDL, and 3) the cholesterol released from the lysosomal hydrolysis of the reconstituted LDL was available to regulate 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesteryl ester formation. The reconstituted LDL was hydrolyzed at a reduced rate by mutant fibroblasts from patients with homozygous familial hypercholesterolemia and by normal fibroblasts that had been grown in 25-hydroxycholesterol plus cholesterol, two conditions in which LDL receptors are known to be decreased. The availability of a method for replacing the endogenous cholesteryl esters of LDL with exogenous neutral lipid should be of value in further studies of the chemistry and metabolism of LDL. Low density lipoprotein, the major cholesterol transport protein in human plasma, consists of an apolar core of neutral lipids surrounded by a polar surface shell (1, 2). The neutral lipid core, which comprises about 50% of the total mass of the particle, consists almost entirely of cholesteryl esters (2), * This research was supported by United States Public Health Service Grant POI-HL from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of United States Public Health Service Postdoctoral Research Fellowship HL chiefly cholesteryl linoleate (3). The polar shell is made up predominantly of phospholipids and unesterified cholesterol molecules. The protein component, designated apoprotein B, is situated in such a way that part of its structure is at the surface and part is buried within the core of the lipoprotein particle (1, 2). A variety of mammalian cells possess on their surfaces receptors that specifically bind plasma LDL with high affinity (reviewed in Refs. 4 and 5). The intact, receptor-bound LDL particle is taken up by the cells through adsorptive endocytosis and delivered to lysosomes where the protein and cho- lesteryl ester components are hydrolyzed. The cholesterol liberated from the hydrolysis of the core cholesteryl esters is utilized by cells for membrane synthesis and also serves to regulate cholesterol metabolism within the cells (4, 5). Compelling genetic and biochemical evidence suggests that the LDL receptor recognizes the protein component of the lipoprotein (6-8). Recently, Shireman et al. (9) showed that a soluble complex of apoprotein B and albumin formed after removing nearly all of the lipid components of LDL retained the ability to bind to the LDL receptor of human fibroblasts. These findings suggested that it might be possible to remove the endogenous cholesteryl ester core of LDL and replace it with exogenous neutral lipids, without altering the ability of the LDL particle to bind to the LDL receptor and be taken up by cells. Such a reconstituted LDL could be valuable as a vehicle for delivering lipophilic compounds to cells that possess LDL receptors. Equally important, the reconstituted LDL particle would facilitate studies of the relation of LDL structure to biological function. In 1965, Gustafson described a method by which the neutral lipid core of human very low density lipoprotein could be extracted selectively into heptane with preservation of a soluble apoprotein phospholipid complex (10). The key step in this procedure was the use of a potato starch powder to stabilize the lipid-depleted apoproteins so that aggregation did not occur during the heptane extraction. Whereas the Gustafson procedure worked well for very low density lipoprotein and high density lipoprotein, the yield of soluble, lipid- Throughout this paper, the term cholesteryl ester refers to the entire cholesteryl ester molecule which contains both cholesterol and a long chain fatty acid. The term esterified cholesterol refers only to the cholesterol component of the cholesteryl ester. The mass of esterified cholesterol in 1 mol of cholesteryl linoleate is 386 g, whereas the mass of cholesteryl linoleate is 649 g. The abbreviations used are: LUL, low density lipoprotein; FH, familial hypercholesterolemia; HMG-CoA reductase, 3-hydroxy-3. methylglutaryl coenzyme A reductase; r-[cl- HILDL, reconstituted low density lipoprotein in which the endogenous free and esterified cholesterol of native LDL was removed by heptane extraction and a soluble particle reconstituted by addition of exogenous [ Hlcholesteryl linoleate. 4093

2 4094 Reconstitution of Biologically Active LDL depleted LDL-protein was only 10% (10). In the current paper, we have utilized the Gustafson procedure to extract the cho- lesteryl esters from the core of LDL, and we subsequently reconstituted the lipid-depleted LDL by adding exogenous [ Hlcholesteryl linoleate in heptane. The resulting reconstituted particle, designated r-[cl- HILDL, is water-soluble and retains many of the physical and biological properties of native LDL. EXPERIMENTAL PROCEDURES Materials-Sodium [ Iliodide (11 to 17 mci/pg), [ 1,2- Hlcholesterol (43 Ci/mmol), and [l-14c]oleic acid (59.5 mci/mmol) were obtained from Amersham/Searle. r>r.-3-hydroxy-3-methy1[3-4cjglutaryl coenzyme A (49.5 mci/mmol) and Aquasol counting fluid were purchased from New England Nuclear Corp. Cholesterol and cholesteryl linoleate were obtained from Applied Science Laboratories Inc. 25.Hydroxycholesterol was purchased from Steraloids. Inc. Chloroquine diphosphate, chicken serum albumin, sucrose, and Tricine (Ntris(hydroxymethyl)methyl glycine) were obtained from Sigma Chemical Co. Linoleoyl chloride was purchased from Nu Check Pren. Inc. Purified potato starch powder (Lot ) was purchased from Fisher Scientific Co. Rabbit anti-human LDL (IgG fraction) and rabbit anti-chicken serum albumin (IgG fraction) were purchased from Cappell Laboratories. Dextran sulfate, sodium salt (M? = 500; 000) was obtained from Pharmacia. Urn-Pore membrane filters (0.8 pm, 25 mm) were purchased from Bio-Rad Laboratories. Other chemicals, thin layer chromatographic supplies, and tissue culture supplies were obtained from sources as previously reported (11-13). Lipoproteins-LDL (density to g/ml) and lioooroteindeficient serum (density > g/ml) were prepared from single 500- ml units of blood collected in 0.1% EDTA from individual healthy subjects who had fasted for 15 h (11). Lipoproteins were fractionated by sequential ultracentrifugation (14) as previously described (11). I-labeled LDL (specific activitv. 200 to 400 cnm/na of nrotein) was prepared as previously described (6). For experiments, ihe 1:LDL was diluted with unlabeled LDL to give the final concentration and specific activity as indicated. Preparation of FHjCholesteryl Linoleate-[ HICholesterol (43 Ci/mmol) was reacted with an excess of linoleoyl chloride in dry pyridine as described by Goodman (15). The resulting [ Hlcholesteryl linoleate was purified and isolated as described by Faust et a/. (16). The final [: H]cholesteryl linoleate was 99% pure as judged by thin layer chromatography on silica gel using benzene-ethyl acetate (2:l) as the developing solvent system and was recovered with a 90% yield. For experiments, the [ HJcholesteryl linoleate was diluted with unlabeled cholesteryl linoleate to give the final concentration and specific activity as indicated. Preparation of Reconstituted LDL-Reconstituted LDL (referred to as r-[cl- H]LDL) was prepared from either native LDL or I- LDL by the following sequential steps: 1) dialysis, 2) lyophilization in the presence of potato starch, 3) extraction of endogenous neutral lipids with heptane, 4) introduction of exogenous [: H]cholesteryl linoleate into the heptane-extracted LDL to form r[cl- H]LDL, and 5) solubilization of the r-[cl- H]LDL in aqueous buffer. Steps 1 to 3 were modified from the lipid extraction method of Gustafson in which a potato starch powder is used to stabilize the LDL-protein during lyophilization and heptane extraction (10). In a typical preparation, 48 mg of native LDL-protein in 15 ml of 0.15 M NaCl and 0.3 mm sodium EDTA, ph 7.4, were dialyzed at 4 C for 60 h against four changes of 6 liters of 0.3 mm sodium EDTA, ph 7.0. Aliquots of the dialyzed LDL (600 ~1 containing 1.9 mg of LDL-protein) were transferred to Siliclad-treated glass tubes (13 X 100 mm) (17), each of which contained 25 mg of purified potato starch. The potato starch was evenly suspended in the LDL solution by agitation with a Vortex mixer, after which each mixture was rapidly frozen along the sides of the tube by immersion in liquid nitrogen. The samples were lyophilized for 6 h and stored overnight at 4 C under vacuum in a desiccator containing P,Os. Next, 5 ml of heptane at -10 C were added to each tube of lyophilized LDL starch complex in order to extract the neutral lipids. After vigorous agitation with a Vortex mixer, each mixture was incubated at -1O C for 1 h with repeated agitation every 10 to 15 min. The lipid-containing heptane supernatant fraction was separated from the starch-ldl residue by centrifugation (2000 rpm, 10 min, 4 C). The starch-ldl residue was subjected to heptane extraction three more times, each extraction involving a 30-min incubation at -10 C. After removal of the last heptane supernatant fraction, 200 ~1 of heptane containing 6 mg of ] H]cholesteryl linoleate (2590 cpm/nmol) were added to each tube. Each mixture was agitated briefly with a Vortex mixer and then incubated at -10 C for 1.5 h. Each tube was then placed in an ice bath and the heptane was evanorated under nitrogen., until the r, Hlcholestervl L _ linoleate/ starch/ldl mixture was powder dry (approximately 30 min). The r- [CL- H]LDL was then solubilized by addition of 1 ml of 10 mm Tricine, ph 8.4, to each tube, followed by incubation at 4 C for 41 h. The soluble r-[cl- H]LDL was separated from the potato starch and the unincorporated ] H]cholesteryl linoleate by centrifugation (2000 rpm, 10 min, 4 C). The supernatant fraction was transferred to a 1.5- ml plastic microfuge tube (Beckman) and subjected to a second centrifugation (12,000 rpm, 20 min, 4 C). The final supernatant fraction contained a small amount of particulate material that tended to float to the top of the solution. This particulate material could be removed by passing the solution through a Uni-Pore membrane filter (0.8 pm) with a 10 to 15% loss of both the protein and cholesteryl ester components of the r-[cl-. H]LDL and no loss of biologic activity. Optimal conditions for preparing r-[cl- H]LDL were determined by varying the following parameters: amount of LDL-protein lyophilized (0.6 mg to 1.9 mg/tube), amount of ] H]cholester.yl linoleate added to the heptane-extracted LDL (0 to 6 mg/tube), salt concentration of the solubilization buffer (0 to 0.5 M NaCl in 10 mm Tricine, ph 8.4), ph of the solubilization buffer (ph 4 to 9.5), and duration of incubation with the solubilization buffer (0 to 108 h) (see Results ). During the course of the experiments reported in this paper, several preparations of r-[cl-. H]LDL were used. Unless otherwise stated, each of these preparations was made using the following conditions: 1.9 mg of LDL-protein/tube, 6 mg of [ Hlcholesteryl linoleate (2590 cpm/nmol)/tube, and a 41-h incubation at 4 C of the ] H]cholesteryl linoleate/starch/ldl mixture in 10 mm Tricine, ph 8.4. Measurement of Cholesterol, Cholesteryl Esters, Triglycerides, and Phospholipids-The content of cholesterol in LDL was measured by a previously described method in which the steroids were extracted with chloroform/methanol (2:1), the free and esterified cholesterol fractions were separated on silicic acid/celite columns and the cholesterol content of each fraction was measured by gasliquid chromatography (following alkaline hydrolysis of the cholesteryl ester fraction) (13). Correction for procedural losses, which averaged 25%. was made by utilizing [ HIcholesterol, cholesteryl [14C]oleate, and stigmasterol as internal standards (13). The relative composition of the fatty acyl components of the cholesteryl esters in LDL was determined by gas-liquid chromatography of the fatty acid methyl esters as previously described (Method 2, Ref. 18) except that the cholesteryl esters were isolated by silicic acid/celite column chromatography and not by thin layer chromatography. The content of phospholipids in LDL was determined bv a previously described method (19) in which the phospholipids were extracted by the method of Folch et al. (20); the lipid-containing lower phase was washed once with pure upper phase solvent, evaporated to dryness, and ashed (21); and the total content of inorganic phosphate was measured by the method of Chen et al. (22). Calculations of phospholipid mass were made assuming that phosphorus accounts for 4% of the total phospholipid weight. The content of triglycerides in LDL was determined by the enzymatic method of Von Schmidt and von Dahl (23) using Boehringer Mannheim triglyceride assay reagents (Reagent Set ). Cells-Cultured fibroblasts were derived from skin biopsies obtained from a normal subject (D.S.) and a patient with the receptornegative form of homozygous familial hypercholesterolemia (M.C.) (24). Cells were grown in monolayer, used between the 5th and 20th passage, and maintained in a humidified incubator (5%) CO,) at 37 C in 75 cm stock flasks containing 10 ml of growth medium consisting of Eagle s minimum essential medium supplemented with penicillin (100 units/ml); streptomycin (100 pg/ml); 20 mm Tricine, ph 7.4; 24 mm NaHCO:!; 1% (v/v) nonessential amino acids; and 10% fetal calf serum. All experiments were performed using a standard format (25). Confluent monolayers of cells from stock flasks were dissociated with 0.05% trypsin, 0.02% EDTA solution. On Day 0, 8 x IO4 fibroblasts were seeded into each 60.mm Petri dish containing 3 ml of growth medium with 10% (v/v) fetal calf serum. On Day 3, the medium was replaced with 3 ml of fresh growth medium containing 10% fetal calf serum. On Day 5, each monolayer was washed with 3 ml of Dulbecco s phosphate-buffered saline (0.8% NaCl solution), after which 2 ml of fresh medium containing 10% (v/v) lipoprotein-deficient serum (final protein concentration, 5 mg/ml) were added. Unless otherwise stated, all experiments were initiated on Day 7 after the cells had been incubated with lipoprotein-deficient serum for 48 h and while the

3 Reconstitution of Biologically Active LDL 4095 cells were in the late phase of logarithmic growth. Hydrolysis of r-[cl- H]LDL by Fibroblast Monolayers-Monolayers were incubated at 37 C with r-[cl- H]LDL in growth medium containing 10% lipoprotein-deficient serum. After the indicated interval, each monolayer was washed as previously described (26), and the cells were harvested and extracted with chloroform/methanol (2:l) after the addition of an internal standard containing [ C]cholesterol (30 pg, 1000 cpm) and unlabeled cholesteryl oleate (30 pg). The free and esterified [ HIcholesterol fractions were separated by thin layer chromatography on silica gel sheets (26) using benzene/ethyl acetate (2:l). Unless otherwise stated, hydrolytic activity is expressed as nanomoles of [: H]cholesterol formed and contained within the cells per mg of total cell protein. The data were corrected for the recovery of [?]cholesterol from each sample, which averaged 85%. Surface Binding, Intracellular Accumulation, and Proteolytic Hydrolysis of I-LDL by Fibroblast Monolayers-Monolayers were incubated at 37 C with I-LDL in growth medium containing 10% lipoprotein-deficient serum (6, 12, 25). After the indicated interval, the medium was removed, the intact I-LDL in the medium was precipitated with 10% (w/v) trichloroacetic acid, the resulting supernatant was extracted with chloroform and hydrogen peroxide to remove free iodine (27), and an aliquot of the aqueous phase was counted to determine the amount of I-labeled (non-iodide) acidsoluble material formed by the cells and released into the medium (12). Hydrolytic activity represents the cell-dependent rate of proteolysis and is expressed as micrograms of I-LDL protein hydrolyzed to acid-soluble material per mg of total cell protein. After removal of the medium, the cell monolayers were washed extensively (25) and then exposed to a solution containing 4 mg/ml of dextran sulfate (25). The I-LDL released from the cell surface by dextran sulfate (releasable I-LDL) represents LDL that was bound at the surface receptor sites. The cells were then harvested by dissolution in 0.1 N NaOH and the amount of I-LDL remaining associated with the cells after dextran sulfate treatment (internalized I-LDL) was measured (25). The total cellular content of I-LDL represents the surface-bound I-LDL plus the internalized I-LDL (12, 25). All of these values are expressed as micrograms of I-LDL protein per mg of total cell protein. Assay of 3-Hydroxy-3-methylglutaryl-CoA Reductase Activity in Cell-free Extracts-The rate of conversion of 3-hydroxy-3-methyl[3- C]glutaryl-CoA (12,000 cpm/nmol) to [ 4C]mevalonate was measured in extracts of detergent-solubilized fibroblasts as previously described (11). HMG-CoA reductase activity is expressed as picomoles of [14C]mevalonate formed per min per mg of detergent-solubilized protein. Incorporation of [l- 4C]Oleate into Cholesteryl Esters by Fibroblast Monolayers-Monolayers were incubated at 37 C with 0.1 rnm [l- 4C]oleate bound to albumin in growth medium containing 10% lipoprotein-deficient serum (28). After the indicated interval, the cells were washed, harvested, and extracted with chloroform/methanol (2:1), and the cholesteryl [14C]oleate was isolated by thin layer chromatography (28). Esterification activity is expressed as picomoles of cholesteryl [?]oleate formed per mg of total cell protein. Correction for procedural losses, which averaged 15%, was made by utilizing vh]cholesteryl oleate as an internal standard added prior to the chloroform/methanol extractions (16). Immunoprecipitations-The LDL immunoprecipitation assay was based on a modification of a method for measurement of very low density lipoprotein as described by Luskey et al. (29). Each immunoprecipitation reaction was performed in a 400~~1 plastic microfuge tube (Beckman) that contained in a final volume of 245 ~1: 10 mm sodium phosphate, ph 7.3; 0.12 M NaCl; 20 pg of protein of r-[cl- H]LDL that was radiolabeled with H in its cholesteryl linoleate component (107,000 cpm) and with? in its protein component (6400 cpm); and 150 pg of anti-ldl (IgG fraction) as indicated. Control precipitation reactions contained 20 pg of chicken albumin plus 150 pg of anti-chicken albumin (IgG fraction) in place of the anti-ldl (29). Each tube was incubated at 37 C for 2 h and allowed to stand overnight at 4 C. The tubes were then centrifuged at 12,000 rpm for 10 min at 4 C in a Beckman microfuge. The supernatant fraction was discarded and the precipitate was washed three times with 300 ~1 of 0.15 M NaCl. The bottom of the tube containing the precipitate was cut off and counted directly in a well-type y counter ( I radioactivity) and then placed in a scintillation vial and counted in 10 ml of Aquasol ( H radioactivity). Density Gradient Centrifugation-Linear gradients of 5 ml containing 5 to 30% (w/v) sucrose in 50 mm Tricine, ph 8.5, were prepared. Samples of r-[cl- H]LDL (0.25 ml in 10 mm Tricine, ph 8.4) were layered on top of the gradient and centrifuged at 40,000 rpm for 36 h at 4 C in a Beckman SW 50.1 rotor. At the end of the centrifugation, the bottom of each tube was punctured, and 5-drop fractions were collected in scintillation vials and counted for radioactivity in 10 ml of Aquasol. To ensure that equilibrium was established during the centrifugation, one sample of I-LDL in,30% sucrose was placed at the bottom of the centrifuge tube prior to addition of the gradient. This sample migrated to the same location in the gradient as did the sample of I-LDL layered on top of the gradient. Sample densities were determined by refractometry. Other Assays-The protein concentration of cell extracts, whole cells, and lipoproteins was determined by the method of Lowry et al. (30) with bovine serum albumin as a standard. Lipoprotein electrophoresis was carried out in agarose gels at ph 8.6 in barbital buffer (31). RESCJLTS When samples of LDL were dialyzed against a dilute EDTA solution, lyophilized in the presence of starch powder, and extracted with heptane, more than 99% of the free and esterified cholesterol was removed (Table I). Most of the protein and phospholipids of the lipoprotein remained associated with the insoluble starch. Exogenous [ Hlcholesteryl linoleate was then introduced into the starch-ldl residue by incubating the residue with a solution of [ Hlcholesteryl linoleate in heptane. The heptane was evaporated and the resulting [ Hlcholesteryl linoleate/starch/ldl mixture was suspended in an aqueous buffer. Upon subsequent centrifugation of the mixture, 56% of the original protein and 24% of the added [ Hlcholesteryl linoleate was found in the supernatant fraction. The relative composition of the material in the supernatant was similar to that of the starting LDL with respect to the proportions of total cholesterol, protein, and phospholipid. However, the supernatant material contained no free cholesterol (Table I). This supernatant material was shown to consist of reconstituted LDL particles and is hereafter desig- nated as r-[cl- HILDL. To follow separately the behavior of the protein and cholesteryl ester components of the reconstituted LDL, the heptane extraction and reconstitution were performed using native LDL that had been mixed with a tracer amount of Y- LDL. Agarose gel electrophoresis of the doubly labeled, reconstituted LDL revealed that the p H]cholesteryl linoleate and I-labeled protein components migrated as a single band with /3 mobility (Fig. 1). Both the [ Hlcholesteryl linoleate and the I-labeled protein components of the r-[cl- H]LDL were precipitated by an antibody to native LDL (Table II). The close association of the cholesteryl linoleate and the protein components of the reconstituted lipoprotein was further confirmed by the observation that more than 97% of both the lz51 and H radioactivity in the r-[cl- H]LDL were precipitated when the reconstituted lipoprotein was exposed to heparin in the presence of manganese, a treatment that quantitatively precipitates native LDL (32). A series of experiments was performed to study the parameters of the reconstitution procedure. Fig. 2 shows the time course of the appearance of LDL-protein and cholesteryl linoleate in the supernatant solution after exposure of the cholesteryl linoleate/starch/ldl mixture to the aqueous buffer. The amount of cholesteryl linoleate in the supernatant fraction increased progressively until it reached a maximum at 5 h. The appearance of protein in the supernatant fraction initially paralleled that of the cholesteryl linoleate. However, after 5 h small amounts of protein continued to appear in the supernatant fraction. In other experiments not shown, we found that increasing the ionic strength of the aqueous buffer by addition of NaCl in concentrations up to 0.5 M did not affect the yield of r-[cl- H]LDL or its chemical or biological properties. When the ph of the solubilization buffer was

4 4096 Reconstitution of Biologically Active LDL TA~I.E Extraction of LDL lipids with heptane Duplicate aliquots of dialyzed I-LDL (1.92 mg of LDL-protein to h at 4 C. Measurements of the total cholesterol, free cholesterol, which a tracer amount of I-LDL (6.3 x lo cpm) was added prior to esterified cholesterol, triglyceride, and phospholipid content of the dialysis) were lyophilized in the presence of starch. Each mixture was indicated samples were determined by chemical methods as described extracted four times with 5 ml of cold heptane and then reconstituted under Experimental Procedures. Protein content was determined with 6 mg of [ Hlcholesteryl linoleate (2590 cpm/nmol) in heptane as either by the Lowry method (Samples A and D) or by measurement described under Experimental Procedures. After evaporation of the of I radioactivity (Samples B, C, and D). Each value represents the heptane, each mixture was incubated in 10 mm Tricine, ph 8.4, for 41 average of duplicate measurements. I Composition Sample A. Untreated I-LDL B. Sequential heptane extracts No. 1 No. 2 No. 3 No. 4 C. Starch-LDL Residue D. r-[cl- H]LDL Protein m&z 1.92 (30.3%) (35.8%) The number in parentheses represents the weight per cent of the indicated component relative to the total mass of the LDL sample. The total mass for Samples A and D were 6.33 and 3.02 mg, respectively. The percentages do not total 100% since the weight of the fatty acid component of the cholesteryl esters is not included (see Footnote 1). The fatty acid composition of the cholesteryl esters of LDL, SLICE NUMBER FIG. 1. Electrophoretic mobility of reconstituted LDL. r-[cl-jh]- LDL was prepared by the standard procedure except that the reconstitution was performed with native LDL to which a tracer amount of I-LDL was added prior to the dialysis (final specific activity of 251- LDL, 320 cpm/ag of protein). Duplicate samples of r-[cl- H]LDL (10 ag of protein; 60,000 cpm of H; 3200 cpm of I) were subjected to electrophoresis in agarose gels (7.2 x 2.5 cm) at ph 8.6 (barbital buffer). After electrophoresis, one gel was fixed and stained with fat red B and the other gel was cut into 16 segments of 4 mm in length for determination of H radioactivity (0) and? radioactivity (0). Slice 1 represents the point of application of the sample. A band of fat red B stain was observed on the fixed gel in a position equivalent to that of Slice 7. varied between 4 and 9.5, a maximal yield of r-[gy3h]ldl was obtained in the ph range from 7 to 9. To study the stoichiometry of the reconstitution reaction, we added varying amounts of [ Hlcholesteryl linoleate to fixed amounts of lyophilized, lipid-depleted LDL-protein (Fig. 3). As the amount of added [ Hlcholesteryl linoleate was increased, the absolute amounts of protein (Fig. 3A) and cholesteryl linoleate (Fig. 3B) in the supernatant solution increased until a plateau was approached. The mass ratio of cholesteryl linoleate to protein also increased with increasing amounts of added cholesteryl linoleate until a maximum was approached (Fig. 3C). At the highest amount of added cholesteryl linoleate (6 mg), the mass ratio of [3H]cholesteryl linoleate to protein in the r-[cl- H]LDL was 1.7 when 0.64 mg of Total Cholesterol Esterified w? % 1.95 (30.8%) (27.2%) looh Triglycerides Pz 0.16 (2.5%) <0.025 (<0.5%) Phospholipids m&y 1.27 (20.1%) 0.56 (18.6%) expressed as the weight per cent of total methylated fatty acids, were as follows: Samule A 16:0. 12%: 16:l. 1%; 18:l. 17%; 18:2. 70%; others. ~1%. Sample D: 18:2, >98%; others, <I%., not determined. This value represents the lower limit of sensitivity of the assay for triglycerides. TARI.E Precipitation of reconstituted LDL by antibody to LDL The r-[cl-: H]LDL was prepared by the standard procedure except that the reconstitution was performed with native LDL to which a small amount of I-LDL had been added prior to the dialysis (final specific activity of I-LDL, 320 cpm/ag of protein). Each immunoprecipitation mixture (245 al) contained 20 ag of protein of r-[cl- H]LDL (107,060 cpm of H, 6400 cpm of lz51) and one of the following: no antibody, anti-ldl, or anti-chicken albumin plus chicken albumin as described under Experimental Procedures. The tubes were incubated, and the immunoprecipitates were washed and processed as described under Experimental Procedures. Each value represents the mean of triplicate assays. Component of reconstituted LDL II Radioactivity in precipitate Anti-chicken al- Minus antibody Anti-LDL bumin + chicken albumin cpm % total CPm % total CPm % total CPm cpm CPm [ HlCholesteryl 724 0:7 106,756 94% 11,ooo 10 linoleate I-labeled pro ,863 92% tein protein was used and the ratio was I.3 when 1.9 mg of protein was used. These ratios are similar to the mass ratio of cholesteryl ester to protein in the starting LDL preparation (1.33) (Table I). As the ratio of cholesteryl linoleate to protein in the reconstituted LDL particle increased, the density of the particle decreased. By varying the amount of added [ Hlcholesteryl linoleate from tracer quantities to 6 mg, we obtained r-[cl- 3H]LDL preparations with cholesteryl 1inoleate:protein ratios varying from nearly 0 to 1.5 (Table III). These reconstituted LDL preparations were subjected to equilibrium density gradient centrifugation (Fig. 4). The r-[cl- H]LDL that was reconstituted with only tracer amounts of [ Hlcholesteryl linoleate (Preparation 1) sedimented near the bottom of the tube at a position corresponding to a density of 1.12 g/ml. Preparation 2, with a cholesteryl 1inoleate:protein ratio of

5 Reconstitution of Biologically Active LDL 4097 OOWTik? FIG. 2. Time course of solubilization of reconstituted LDL. Aliquots of dialyzed LDL (662 ag of protein) were lyophilized in the presence of starch, the neutral lipids were extracted, and each sample was reconstituted with 2.6 mg of [: HJcholesteryl linoleate (178,000 cpm/ag) in heptane as described under Experimental Procedures. After evaporation of the heptane, each mixture was incubated in 10 mm Tricine, ph 8.4, at 4 C for the indicated time. The supernatant fraction was isolated by centrifugation and its content of cholesteryl linoleate (0) and protein (0) was determined by scintillation counting and the Lowry method, respectively. Each value represents the average of duplicate samples. A Protein Recovered Hours $ 0 I 0 q 8 Cholesteryl Linoleate Recovered Linoleote to Protein [3ti] Cholesteryl Llnoleote Added (mg) FIG. 3. Formation of reconstituted LDL: effect of increasing amounts of cholesteryl linoleate added to two different amounts of LDL-urotein. Aliauots of dialvzed LDL containing 0.64 me (0) or 1.9 mg (A) of LDL-protein were lyophilized in the presence if.2; mg of starch, the neutral lipids were extracted, and each sample was incubated with the indicated amount of c H]cholesteryl linoleate in heptane as described under Experimental Procedures. Each tube contained the same amount of H radioactivity (4.5 x 10 cpm) with varying specific activity. After evaporation of the heptane, each mixture was incubated for 18 h at 4 C in 10 mm Tricine, ph 8.4. The supernatant fraction was isolated by centrifugation and its content of protein (A) and cholesteryl linoleate (B) was determined by the Lowry method and scintillation counting, respectively. The mass ratio of cholesteryl linoleate to protein in the supernatant fraction is shown in C. Each value represents the average of duplicate samples. 0.13, was heterogeneous in density with its major component being intermediate in density between Preparation 1 and the a51-ldl that was used as a standard (density, g/ml). The two r-[cl- H]LDL preparations with cholesteryl lino- 1eate:protein ratios of 0.71 and 1.5 (Preparations 3 and 4) exhibited densities slightly greater than that of the I-LDL. The density of the peak fraction in Preparation 4 (cholesteryl 1inoleate:protein ratio of 1.5) was g/ml. Tracer amounts of C Hlcholesteryl linoleate placed on the top of the gradient in aqueous suspension in the absence of protein did not enter the gradient. In an initial test for preservation of the biological activity of the reconstituted LDL, we compared native LDL and reconstituted LDL for their ability to compete with I-LDL for binding, uptake, and degradation by fibroblasts at 37 C. The data in Fig. 5 indicate that the native LDL and the r-[cl- HJLDL were of equal potency in their ability to compete with I-LDL, as judged by the similar shapes of the competition curves for all three receptor-mediated processes. To compare directly the metabolism of I-LDL and r-[cl- HILDL, we incubated each lipoprotein preparation with fibroblasts and measured the uptake and hydrolysis as a function of time (Fig. 6). In the case of I-LDL the cellular content of radioactivity reached a steady state plateau by 1 h (Fig. 6A). Previous studies have shown that this radioactivity within the cell represents relatively intact trichloroacetic acidprecipitable?-ldl that is in the process of being hydrolyzed (12). Nearly all of the I-LDL that was hydrolyzed by the cells appeared in the medium as trichloroacetic acid-soluble radioactivity (Fig. 6A). The rate of appearance of the trichloroacetic acid-soluble material was linear with time for 6 h after a brief initial lag. The metabolism of the r-[cl- H]LDL was generally similar to that of the I-LDL (Fig. 6B). Thus, the cellular content of intact, unhydrolyzed [ Hlcholesteryl linoleate rose rapidly and reached a plateau within 1 h. After a brief lag phase, free [ HIcholesterol began to appear in the cell, and this accumulation continued in a linear fashion for 6 h. At the end of this interval, the ratio of free [ HIcholesterol to intact [ Hlcholesteryl linoleate in the cell was 4.3. This ratio was slightly less than the ratio of trichloroacetic acidsoluble I radioactivity to intact cellular I-LDL protein (6.3) (Fig. 6A). By adding together the values for intact [ HIcholesteryl linoleate and free [ HIcholesterol in the cells, we calculated that at 6 h a total of 8.4 nmol of [ Hlcholesteryl linoleate had been taken up and hydrolyzed by the cells per mg of cell protein. Inasmuch as the ratio of the nanomoles of cholesteryl linoleate to micrograms of protein in the r-[cl- H]LDL was 2.0 (Table I), the data in Fig. 6B indicate that at 6 h a total of 4.2 pg of LDL-protein in the r-[cl- H]LDL would have been taken up and hydrolyzed if the LDL particle were taken up intact. This value agrees fairly closely with the measured value for total uptake and hydrolysis of LDL protein using I-LDL (4.9 fig) (Fig. 6A). Thus, the r-[cl- H]LDL and the I-LDL were both taken up and hydrolyzed in a similar fashion in fibroblasts. However, whereas all of the Ilabeled acid-soluble material that was formed by the cells was excreted into culture medium (la), most of the [ HIcholesterol formed was retained by the cells (see below). Fig. 7 compares the uptake and hydrolysis of I-LDL and r-[cl- H]LDL as a function of the concentration of lipoprotein added to the culture medium. The similarity in shapes of the saturation curves for both preparations suggests that the uptake of r-[cl- HILDL, like that of I-LDL, is receptormediated. Double reciprocal plots revealed that the concentrations of 1-LDL and r-[cl- H]LDL giving half-maximal rates of hydrolysis were 17 and 21 pg of protein/ml, respectively. To confirm that the r-[cl- H]LDL was being taken up by the LDL receptor and hydrolyzed within lysosomes, we performed the experiment shown in Table IV. In this experiment, the hydrolysis of r-[cl- H]LDL and I-LDL were both found

6 4098 Reconstitution of Biologically Active LDL TABI,F, Characteristics of reconstituted LDL preparations used for density gradient centrifugation Aliquots of dialyzed LDL (620 pg of protein) were lyophilized in isolated by centrifugation and its content of protein and cholesteryl the presence of starch, the neutral lipids were extracted, and each linoleate was determined by the Lowry method and scintillation sample was reconstituted with of heptane containing the counting, respectively. Each of the preparations of r-[cl- H]LDL indicated amount of [3H]cholesteryl linoleate (6 X 10 cpm/tube). was further analyzed by sucrose gradient centrifugation as shown in After evaporation of the heptane, the mixtures were incubated in 10 Fig. 4. mm Tricine, ph 8.4, for 85 h at 4 C. The supernatant fraction was tion No. III Amount added Amount recovered in r-[cl- H]LDL r-[cl- H]LDI, applied to sucrose gradient LDL pro- ~ H]Cholesteryl tein linoleate Protein [: H]Cholesteryl linoleate Mass ratio of [ HIcholesteryl linoleate to protein Protein ~ H]Cholesteryl linoleate!x M!@/!Jl?!A? CPm (a) (b) (b)/(a) ,GQo , , ,000 FRACTION NUMBER FIG. 4. Density gradient centrifugation of lipid-extracted LDL preparations that were reconstituted with varying amounts of [ HIcholesteryl linoleate. Each linear sucrose density gradient (5 to 30% sucrose in 50 mm Tricine, ph 8.5) was overlayered with 250 ~1 of 10 mm Tricine, ph 8.4, containing one of the following:?-ldl (3 pg, 860,000 cpm), [ Hlcholesteryl linoleate (48 pmol, 750,006 cpm), or one of the four r-[cl- H]LDL preparations described in Table III. Each gradient was spun for 36 h at 40,000 rpm in a SW 50.1 rotor at 4 C. Fractions (5 drops each) were collected and counted for radioactivity as described under Experimental Procedures. The recovery of H radioactivity in the four r-[cl- H]LDL preparations after centrifugation ranged from 90 to 98%. The data are expressed as the per cent of radioactivity in each fraction relative to the total radioactivity recovered in the gradient: H, [ Hlcholesteryl linoleate; A---A, I-LDL; U, r-[cl- HILDL, No. 1; 0. 0, No. 2; 0-0, No. 3; A-A, No. 4. Fraction No. 1 represents the bottom of the gradient. : A WL d? Q Y k! c A. RECEPTOR BINDING :\ a\ \e 0. \ 0 8 f/ B. INTERNALIZATION \ \ f/ I t C. HYDROLYSIS 8\ Addifion III to Medium to be reduced in cells from an FH homozygote that lack LDL receptors. Moreover, in normal cells the hydrolysis of the protein and cholesteryl esters was reduced to the same degree by the addition of excess unlabeled LDL which competes for binding to the LDL receptor. Finally, the addition of chloroquine, an inhibitor of lysosomal LDL degradation (18, 26,33), blocked the hydrolysis of both the cholesteryl linoleate component of r-[cl- H]LDL and the protein component of rz51- LDL in the normal cells. The hydrolysis of r-[cl- H]LDL and?-ldl were also shown to be reduced proportionately when fibroblasts were grown in the presence of a mixture of 25-hydroxycholesterol plus cholesterol (Table V). This sterol mixture has been shown COMPETING LIPOPROTEIN (pg protein/ml) FIG. 5. Ability of reconstituted LDL to compete with I-LDL for binding, internalization, and proteolytic hydrolysis in fibroblasts at 37 C. On Day 7 of cell growth, each monolayer of normal cells received 2 ml of growth medium containing 10% lipoprotein-deficient serum, 25 pg of protein/ml of la5 I-LDL (126,000 cpm/pg of protein), and the indicated concentration of either unlabeled LDL (0) or r- [CL- HJLDL (2590 cpm/nmol of cholesteryl linoleate) (0). After incubation for 5 h at 37 C, the amount of dextran sulfate-releasable I-LDL (receptor-bound), dextran sulfate-resistant I-LDL (internalized), and?-ldl hydrolyzed were determined as described under Experimental Procedures. Each value represents the average of duplicate incubations.

7 Reconstitution of Biologically Active LDL t A 251-LDL % HOURS FIG. 6. Time course of uptake and hydrolysis of I-LDL and reconstituted LDL. On Day 7 of cell growth, each monolayer of normal cells received 2 ml of medium containing 10% lipoproteindeficient serum and the dishes were divided into two sets. Each of the monolayers in one set of dishes received 10 pg/ml of I-LDL (107,090 cpm/pg of protein) and each of the monolayers in the other set received 10 pg/ml of r-[cl- H]LDL (2590 cpm/nmol of cholesteryl linoleate). Both sets were incubated at 37 C for the indicated time. The additions of I-LDL and r-[cl- H]LDL were arranged in such a way that all of the monolayers were harvested at the same time. For A, the total cellular content of I-LDL (0) and the total amount of I-LDL hydrolyzed and excreted into the medium (W) were determined as described under Experimental Procedures. For B, the total cellular content of [ Hlcholesteryl linoleate (0) and the total amount of [ HIcholesterol formed from the hydrolysis of r-[cl- H]- LDL and contained within the cells (A) were determined as described under Exnerimental Procedures. Each value renresents the average of duplicat e incubations. 2. A. Cellular Content I LDL (pg protein/ml) FIG. 7. Saturation kinetics for the uptake (A) and hydrolysis (B) of reconstituted LDL (0) and I-LDL (A) by fibroblasts at 37 C. On Day 7 of cell growth, each monolayer of normal cells received 2 ml of medium containing 10% lipoprotein-deficient serum and the indicated concentration of either r-[cl- H]LDL (2590 cpm/nmol of cholesteryl linoleate (CL)) or I-LDL (54,000 cpm/pg of protein). After incubation for 6 h at 37 C, the total cellular content of [ Hlcholesteryl linoleate (0) and lt51-ldl (A) were determined (A). The total amount of [ HIcholesterol formed from the hydrolysis of r-[cl- H]LDL and contained within the cells (0) and the total amount of I-LDL hydrolyzed and excreted into the medium (A) were also determined (B). Each value represents the average of duplicate incubations TABLE Hydrolysis of r-[cl- H]LDL and I-LDL by normal and FH homozygote fibroblasts in absence and presence of chloroquine and unlabeled LDL On Day 7 of cell growth, each monolayer received 2 ml of growth medium containing 10% lipoprotein-deficient serum, either 10 pg of protein/ml of r-[cl-. H]LDL (2590 cpm/nmol of cholesteryl linoleate) or 10 pg of protein/ml of I-LDL (144,000 cpm/pg of protein) and the indicated addition. After incubation for 6 h at 37 C, the amount of vh]cholesteryl linoleate hydrolyzed and the amount of I-LDL hydrolyzed were determined as described under Experimental Procedures. Each value reoresents the average of duolicate incubations. Addition medium to None Unlabeled LDL (460 pg protein/ml) Chloroquine (75 PM) Unlabeled LDL + chloroquine IV [ H]Cholesteryl linoleak hydrolyzed I-LDL hydrolyzed Normal FH homozv- Normal FH homozv cells gate cells gote- U?llS cells mm16 h- II@ pg6h- mg TABLE V Parallel reduction in rates of hydrolysis of r-[cl- H/LDL and lz51- LDL in fibroblasts incubated with sterols On Day 5 of cell growth, the medium was replaced with 2 ml of growth medium containing 10% lipoprotein-deficient serum and 3 ~1 of ethanol with or without of 25-hydroxycholesterol and 25 pg of cholesterol. After 48 h at 37 C (Day 7), each dish received either 10 pg of protein/ml of r-[cl- H]LDL (2590 cpm/nmol of cholesteryl linoleate) or 10 pg of protein/ml of I-LDL (116,000 cpm/pg of protein) in the absence or presence of 500 pg of protein/ml of unlabeled LDL. After incubation for 6 h at 37 C, the amount of [ Hlcholesteryl linoleate hydrolyzed and the amount of I-LDL hydrolyzed were determined as described under Experimental Procedures. Each value represents the average of duplicate incubations. The high affinity values represent the difference between the value obtained in the absence of unlabeled LDL (total value) and the value obtained in the presence of excess unlabeled LDL. Prior treatment [~ H]Cholesterol linoleate hydrolyzed I-LDL hydrolyzed of cells Total Hieh affktv Total Hieh affinitv mm16 h- mg- p+y6h-1mg-i Ethanol (100%) (100%) 25-Hydroxycholes (23%) (19%) terol + cholesterol n The number in parentheses represents the high affinity value expressed as a percentage of the high affinity value in cells incubated without sterols. previously to cause a specific decrease in the number of LDL receptors (34). In measuring the hydrolysis of r-[cl- H]LDL by fibroblasts, we discarded the incubation medium and measured only the free [ HIcholesterol that accumulated within the cells. This technique lowered the blank value for the assay by eliminating the necessity to spot large amounts of unhydrolyzed [ Hlcholesteryl linoleate from the medium on the thin layer sheets. In control experiments, we observed that small amounts of free [ HIcholesterol accumulated in the medium during the standard 6-h incubation, This accumulation was 10 to 15% of the total amount of free [ HIcholesterol formed during the incubation. Unlike the free [ HIcholesterol formedand retained by the cells, the generation of the free [ HIcholesterol in the incubation medium did not require the action of the LDL receptor for the following reasons: 1) it was not reduced by chloroquine, 2) it was not reduced by prior

8 4100 Reconstitution of Biologically Active LDL incubation of the cells with 2Shydroxycholesterol plus cholesterol, and 3) it was similar in cells from normal subjects and subjects with homozygous FH. Moreover, when the r- [CL- H]LDL was incubated for 6 h at 37 C in medium that had been conditioned by prior incubation with normal fibroblasts for 48 h, free [ HIcholesterol was formed even in the absence of cells. Inasmuch as fibroblasts have previously been shown to release a lysosomal cholesteryl ester hydrolase into the culture medium (35), we believe that the relatively small amounts of free [ HIcholesterol that appeared in the medium during the 6-h incubation may have been formed by the action of this excreted lysosomal enzyme. The free cholesterol that was liberated from the receptormediated intracellular hydrolysis of the cholesteryl esters of the reconstituted LDL was able to regulate cellular cholesterol metabolism. Fig. 8 shows that the r-[cl- H]LDL was able to suppress HMG-CoA reductase in a fashion similar to that of native LDL. Table VI shows that this suppression did not occur in cells from an FH homozygote, again confirming the requirement for the LDL receptor in mediating the uptake of r-[cl- H]LDL (11). In the experiment shown in Table VII, the ability of r-[cl- H]LDL and native LDL to enhance the rate of incorporation of [ 4C]oleate into cholesteryl [ 4C]oleate was compared. This enhanced cholesterol esterification activity has been shown previously to result from receptor-mediated uptake of LDL and to require hydrolysis of its cholesteryl ester component (18, 28). The data show that r-[cl- H]LDL, like native LDL, caused a marked stimulation in the rate of cholesteryl ester synthesis by the normal fibroblasts. This response did not occur in the FH homozygote cells, again indicating a requirement for the LDL receptor (28). E--J 1 Addition to Medium I I I I I LDL (: 9 protein/ml) Fro. 8. Suppression of HMG-CoA reductase activity in fibroblasts by reconstituted LDL. On Day 7 of cell growth, each monolayer of normal cells received 2 ml of growth medium containing 10 S lipoprotein-deficient serum and the indicated concentration of either native LDL (0) or r-[cl- H]LDL (2590 cpm/nmol of cholesteryl linoleate) (0). After incubation for 6 h at 37 C, the cells were harvested for measurement of HMG-CoA reductase activity. Each value represents the average of duplicate incubations. T~nrx Lack of suppression of HMG-CoA reductase activity by reconstituted LDL in FH homozygote fibroblasts On Day 7 of cell growth, each monolayer received 1.5 ml of growth medium containing 10% lipoprotein-deficient serum and either no addition or 22 pg of protein/ml of r-[cl- HILDL. After incubation for 6 h at 37 C, the cells were harvested for measurement of HMG- CoA reductase activity. Each value represents the average of duplicate incubations. Addition None r-tcl- HlLDL to medium VI HMG-CoA reductase activitv Normal cells FH homozygote cells pm01 min- mgml TAHI.E Effect of reconstituted LDL on rate of incorporation of ( 4CJoleate into cholesteryl [ 4C]oleate in normal and FH homozygote fibroblasts On Day 7 of cell growth, each monolayer received 1.5 ml of growth medium containing 10% lipoprotein-deficient serum and 22 pg of protein/ml of the indicated lipoprotein. After incubation for 5 h at 37 C, each monolayer was pulse-labeled for 2 h at 37 C with 0.1 rnm [?]oleate-albumin (11,700 cpm/nmol), after which the cells were harvested for measurement of their content of cholesteryl [Wloleate. Each value represents the average of duplicate incubations. [ C]Oleate + Cholesteryl rwqoieate Addition None Native LDL r-[cl- H]LDL to medium DISCUSSION VII Normal cells FH homozygote WllS pm01 h- mg In the current studies, we describe a procedure by which more than 99% of the core cholesteryl esters were removed from human LDL and the lipoprotein was subsequently reconstituted by the addition of exogenous neutral lipid. The endogenous cholesteryl esters were removed by heptane extraction in the presence of starch as described by Gustafson (10). The subsequent addition of cholesteryl linoleate in heptane, followed by evaporation of the heptane, led to the incorporation of the exogenously added cholesteryl linoleate into a water-soluble, reconstituted LDL particle. As the mass of cholesteryl linoleate added to a fixed mass of LDL-protein was increased, the cholesteryl 1inoleate:protein ratio in the reconstituted LDL increased until it approached a maximum at a value of approximately 1.5. Inasmuch as this value is similar to the cholesteryl ester:protein ratio of native LDL (3), these data suggest that the physical properties of the protein and phospholipid of LDL limit the amount of cholesteryl ester that can be accommodated in the lipoprotein. For biological studies, we used a standard set of conditions that gave reconstituted LDL preparations with cholesteryl 1inoleate:protein mass ratios of 1.3 to 1.5. In four different experiments in which the same standard conditions were used, the recovery of protein in the soluble reconstituted LDL ranged from 52 to 56%. In total, we have performed the reconstitution procedure more than 60 times using various modifications of the standard procedure. Although the yields of LDL-protein in the r-[cz3h]ldl varied according to the parameter that was varied, each preparation of reconstituted LDL was metabolized by the LDL pathway of fibroblasts in a manner similar to that of native LDL. The chemical composition of the reconstituted LDL prepared by the standard procedure resembled that of native LDL with two major exceptions: 1) all of the cholesteryl esters consisted of cholesteryl linoleate instead of the usual mixture of cholesteryl esters in native LDL, and 2) unesterified cholesterol, which makes up about 7% of the mass of native LDL (l-3), was not present in the reconstituted LDL. Despite these chemical differences, the reconstituted LDL retained its p mobility on agarose gel electrophoresis and its ability to be precipitated by an antibody to native LDL and by heparin/manganese. The mean density of the reconstituted LDL (1.042 g/ml) was slightly higher than that of native LDL (1.034 g/ml). The availability of the LDL receptor system in human fibroblasts enabled us to monitor the biological activity of the reconstituted LDL. These studies revealed not only that the reconstituted LDL could bind to the receptor with the same affinity as that of native LDL, but also that the particle was