Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein.

Abstract

Selective radioactive labeling of the cholesteryl esters contained within human plasma low density lipoprotein has allowed the study of the metabolism of these molecules in monolayers and extracts of cultured human fibroblasts. In monolayers of normal cells, binding of low density lipoprotein to its cell surface rece tor was followed by rapid hydrolysis of the [3H] METHODS Cells. Cultured fibroblasts were derived from skin biopsies obtained from either normal subjects or patients with the receptor-negative form of homozygous familial hypercholesterolemia (10). All cells were grown in monolayer and used between the 5th and 20th passage. Cells were maintained in a humidified incubator (5% CO2) at 370 in 75 cm2 stock flasks (Falcon) containing 10 ml of growth medium consisting of Eagle's minimum essential medium (Gibco, Cat. no. F-il) supplemented with penicillin (100 units/ml); streptomycin (100 ug/ml); 20 mM Tricine-Cl, pH 7.4; 24 mM NaHCO3; 1% (v/v) nonessential amino acids; and 10% (v/v) fetal calf serum (Flow Laboratories). All experiments were done in a similar format: Confluent monolayers of cells from stock flasks were dissociated with 0.05% trypsin/0.02% EDTA solution and were seeded (day 0) at a concentration of 1 X 105 cells per dish into 60 X 15 mm dishes (Falcon) containing 3 ml of growth medium with 10% fetal calf serum. On day 3 the medium was replaced with 3 ml of fresh growth medium containing 10% fetal calf serum. On day 6, when the cells were not yet confluent, each monolayer was washed with 3 ml of Dulbecco's phosphate-buffered saline, and 2 ml of fresh medium containing 5% (v/v) human lipoprotein-deficient serum (LPDS) was added (final protein concentration 2.5 mg/ml). All experiments were initiated on day 7 after the cells had been incubated for 24 hr in the presence of LPDS.Lipoproteins. Human LDL (density 1.019-1.063 g/ml) and LPDS (density > 1.215 g/ml) were obtained from the 2926 Biochemistry: Brown et al.plasma of healthy subjects and prepared by differential ultracentrifugation (5). In the LDL used in the present studies, the ratio of total cholesterol to protein content on a weight basis was 1.6:1, and 70% of the total cholesterol was esterified. Cholesteryl linoleate comprised 50% of the total cholesteryl esters and cholesteryl oleate comprised 20%.Preparation chloroform/methanol (2:1) was evaporated to dryness and taken up in 0.56 ml of solution containing 0.15 M NaCl; 0.3 mM EDTA, pH 7; and 10% (v/v) dimethylsulfoxide. The mixture was incubated for 10 min at 370, after which 10 ,g of LDL (300-400 14g of protein) were added and the incubation was continued for 4 hr at 370. The solution was then dialyzed overnight at 40 against 6 liters of buffer containing 0.15 M NaCl and 0.3 mM EDTA, pH 7, after which it was centrifuged in a Beckman Microfuge (12,000 rpm, 5 min, 240). The supernatant solution, which routinely contained 7-12% of the starting radioactivity, was stored at 40 and used for assays within 1 week. The specific activity of cholesteryl linoleate in the [3H]CL-LDL was d...

Association of cholesteryl ester transfer protein with HDL particles reduces its proteolytic inactivation by mast cell chymase

“…The HDL 3 preparation used in this study contained 0-1.5% of preb-migrating species. LDL was acetylated in the presence of acetic anhydride (32), and the acetyl-LDL was radiolabeled by treatment with [1,2-3 H]cholesteryl oleate (Amersham Biosciences, Piscataway, NJ) dissolved in dimethyl sulfoxide (33). The specific activity of the [ 3 H]CE-acetyl-LDL preparation was 46 dpm/ng protein.…”

Metabolism of acetylated low density lipoproteins by monocyte-derived macrophages from patients with Werner's syndrome.

“…The upper layer was further subjected to zonal ultracentrifugation, and the 0-VLDL was separated. LDL was acetylated by the method of Basu et al 18 Acetylated LDL was iodinated with sodium by using dimethyl sulfoxide and the method of Brown et al 18 The specific activity of 12S l-acetylated LDL was 69 cpm/ ng protein, and the percent lipid labeling was less than 4.5%. The specific activities of 3 H-cholesteryi linoleatelabeled acetylated LDL fH-CE-acetylated LDL) and 3 H-cholestery1 linoleate-labeled 0-VLDL fH-CE-jS-VLDL) were 26.2 and 21.3 dpm/ng total cholesterol, respectively.…”

“…Then, the degradation and cellular association of 12S l-acetyiated LDL were determined by the method of Goldstein and Brown. 18 Briefly, the nontodide radioactivity in the trichloroacetJc acid-soluble fraction of the medium was counted to determine the degradation of 126 l-acetylated LDL. Additional incubation of 125 l-acetylated LDL without cells was used to correct for non-cell-mediated degradation, 20 which was less than 10% of the total degradation.…”

Hydrolysis of Lipid Droplets in Acid Cholesteryl-Esterase-Deficient Fibroblasts.

“…Ultrastructural observations have shown that the lipids accumulate in cells in two distinct states (6,9,23,24), either as lipid droplets surrounded by a limiting membrane in the phagolysosomesor as droplets without a limiting membranein the cytoplasm. Cholesteryl ester-lipid droplets are originally derived from lipoproteins in the blood stream (2,3). Arterial cells take up lipoproteins by endocytosis, hydrolyze the cholesteryl esters with lysosomal acid cholesteryl esterase, and re-esterify the free cholesterol with microsomal acyl-CoA: cholesterol acyltransferase (ACAT) (4, 5, 10).…”