/. Biochem., 80, 729-733 (1976)
Effect of Cholesterol Sulfate and Sodium Dodecyl Sulfate on Lecithin-cholesterol Acyltransferase in Human Plasma Mitsuo NAKAGAWA and Shoji KOJIMA Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Kumamoto 862 Received for publication, March 31, 1976
The effects of cholesterol sulfate and sodium dodecyl sulfate (SDS) on the esterification of cholesterol in sonicated dispersions of lecithin-cholesterol mixtures by lecithincholesterol acyltransferase [EC 2.3.1.43] (LCAT) in human plasma were studied in vitro. The acyltransferase activity was inhibited at concentrations of cholesterol sulfate higher than 1 x 10~4 M. This inhibition was not eliminated by the addition of bovine serum albumin or CaCli. On the contrary, the acyltransferase activity was stimulated at concentrations of SDS ranging from lXlO" 5 M to lXlO" 8 M, and maximum stimulation was obtained at 5xlO~* M. The maximum stimulation disappeared on the addition of bovine serum albumin (30 mg per ml of incubation medium), 1x10"' M CaCl2 or lxlO~* M cholesterol sulfate. On the other hand, the extent of inhibition of the acyltransferase by cholesterol sulfate was not affected by the amount of lecithin in the dispersion added as a substrate, but the maximum stimulation (5xlO~4 M SDS) of the acyltransferase was interfered with when a large amount of lecithin was present in the dispersion. In addition, the amount of SDS required for maximum cholesterol esterification was not affected by the amount of lecithin present in the dispersion. These results suggest that the action of cholesterol sulfate on the acyltransferase is different from that of SDS.
Human plasma contains lecithin-cholesterol acyltransferase [EC 2. 3.1.43] (LCAT), which catalyzes the transfer of an acyl group from the /3-position of lecithin to cholesterol (7). The acyltransferase is inhibited by cholesterol ester and lysolecithin, which are the endproducts of this reaction {2, 3). On the other hand, it has been reported that cholesterol is converted to cholesterol sulfate by the transfer of a sulfonyl group from ascorbic 2-sulfate to cholesterol in mammalian Vol. 80, No. 4, 1976
tissues (4). Recently, it has been found that cholesterol sulfate is a normal constituent of human erythrocytes and plasma and has a protective action against hypotonic hemolysis of human blood cells as an amphipathic lipid (5). However, its biological function is not clear, In this paper, the effects of cholesterol sulfate and SDS, a well-known surfactant, on LCAT in human plasma were compared,
729
730
M. NAKAGAWA and S. KOJIMA
tubes, flushed with N t , sealed and incubated at 37° for 3 hr with constant shaking. After MATERIALS AND METHODS incubation, extraction and separation of lipids Preparation of the Enzyme—As an enzyme and measurement of the radioactivity were source for LCAT, human plasma was obtaine'd conducted as described previously (6~). The] from outdated human blood containing 0.15 determipation of free cholesterol in human volume of anticoagulant solution (citric acid, plasma 'was performed by the procedure desodium citrate, and glucose) by centrifigation scribed by Muesing and Nishida (9). The and was then dialyzed against Tris-HCl buffer, protein content was determined by the procedure described by Lowry et al. (10) with pH 7.0, ionic strength 0.1. Compounds—Cholesterol and SDS were ob- crystalline bovine serum albumin as a standard. tained from Kanto Chemical Co. (Tokyo, Japan). [3H]Cholesterol was purchased from RESULTS AND DISCUSSION New England Nuclear Corp. (Boston, Mass., U.S.A.) and purified as described previously We have previously reported that when soni(6). Bovine serum albumin was purchased cated dispersions of lecithin and cholesterol from Sigma Chemical Co. (St. Louis, Mo., mixtures are used as substrates for LCAT in U.S.A.). Lecithin was prepared from egg yolk human plasma, cholesterol esterification inby the method of Faure (7) and purified by creases linearly with time up to 3 hr (11). Therefore, using similar experimental condisilicic acid column chromatography. Thintions, the effect of cholesterol sulfate on the layer chromatography of the purified lecithin acyltransferase was compared with that of on silica gel G plates with chloroform-methSDS, which is a well-known amphipathic and anol-water (65 : 25 : 4, v/v) as a developing surface-active compound. As shown in Fig. 1, solvent gave a single spot of lecithin. Potassium cholesterol sulfate was synthesized chemically and purified by the procedure of Mumma (8). Preparation of Substrate Dispersion—Sonicated dispersions of lecithin and cholesterol mixtures were prepared as described previously (6). We confirmed by thin-layer chromatography that no degradation of lecithin to lysolecithin occurred during sonication. The lecithin/cholesterol molar ratios of the dispersions used were 2.2, 5.4, and 6.2. The radioactivity and amount of free cholesterol in dispersions having molar ratios of 5.4 and 6.2, and 2.2 were 0.2 ^Ci/0.16 /imole/ml incubation medium 0 10" 5 10~4 10" 1 CHOLESTEROL SULFATE OR SDS (M) and 0.2 ^Ci/0.13 /^mole/ml incubation medium, respectively. The determination of cholesterol Fig. 1. Effect of cholesterol sulfate (curves 3 and 4) and phospholipid phosphorus contents was per- and SDS (curves 1 and 2) on cholesterol esterification in the presence (curves 2 and 4) or absencs (curves formed as described previously (fJ). Enzyme Assay — The incubation mixture 1 and 3) of bovine serum albumin (30 mg/ml). The incubation medium contained 0.1 ml of the dispersion contained 0.1 ml of the dispersion and 0.2 ml (lecithin/cholesterol molar ratio of 6.2), 0.2 ml of of human plasma. The final volume was ad- human plasma and various amounts of cholesterol justed to 0.5 ml with Tris-HCl buffer, pH 7.0, sulfate or SDS. The final volume was adjusted to ionic strength 0.1. Human plasma used for 0.5 ml with Tiis-HCl buffer and the mixture was the incubation was diluted with Tris-HCl buffer incubated at 37° for 3 hr. The percentage of radioto give a protein content of 60 mg per ml. active cholesterol esterified during incubation is given The samples were placed in 15 ml screw-capped on the ordinate. / . Biochem.
AMPHIPATHIC MOLECULES AFFECTING LCAT
the'acyltransferase activity was" depressed at concentrations of cholesterol sulfate • higher than 1 X 1 0 ~ 4 M (curve 3) and decreased to approximately 79%, 27%, and 23% of the control level on the addition of lxlO~ 4 M, 5xlO~ 4 M, and 1X 10~8 M (467 fig per ml of incubation medium) cholesterol sulfate, respectively. The inhibitory effect of cholesterol sulfate reached a plateau at a concentration of approximately l x l O - ' M cholesterol sulfate. The concentration of cholesterol sulfate required for 50% inhibition of the acyltransferase was approximately 3 X 10~4 M (140 fig per ml of incubation medium). Since Bleau et al. (5) have reported that approximately 360 fig of cholesterol sulfate is contained per 100 ml of human plasma as a normal constituent, the cholesterol sulfate concentration required for 50% inhibition was approximately 40 times greater than the concentration in human plasma. However, assuming that the cholesterol in human plasma reached equilibrium with the radioactive cholesterol in the substrate dispersion during incubation, the cholesterol sulfate concentration required for 50% inhibition corresponds to approximately two-thirds of the amount of free cholesterol (approximately 221 fig per ml of incubation medium) in the incubation medium with the dispersion and human plasma which contained approximately 390 pg cholesterol per ml of plasma. Therefore, although the mechanism of inhibitory action of cholesterol sulfate on the acyltransferase is not clear, the interaction of cholesterol sulfate with the enzyme or high density lipoprotein (1.125
Effect of Cholesterol Sulfate and Sodium Dodecyl Sulfate on Lecithin-cholesterol Acyltransferase in Human Plasma Mitsuo NAKAGAWA and Shoji KOJIMA Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Kumamoto 862 Received for publication, March 31, 1976
The effects of cholesterol sulfate and sodium dodecyl sulfate (SDS) on the esterification of cholesterol in sonicated dispersions of lecithin-cholesterol mixtures by lecithincholesterol acyltransferase [EC 2.3.1.43] (LCAT) in human plasma were studied in vitro. The acyltransferase activity was inhibited at concentrations of cholesterol sulfate higher than 1 x 10~4 M. This inhibition was not eliminated by the addition of bovine serum albumin or CaCli. On the contrary, the acyltransferase activity was stimulated at concentrations of SDS ranging from lXlO" 5 M to lXlO" 8 M, and maximum stimulation was obtained at 5xlO~* M. The maximum stimulation disappeared on the addition of bovine serum albumin (30 mg per ml of incubation medium), 1x10"' M CaCl2 or lxlO~* M cholesterol sulfate. On the other hand, the extent of inhibition of the acyltransferase by cholesterol sulfate was not affected by the amount of lecithin in the dispersion added as a substrate, but the maximum stimulation (5xlO~4 M SDS) of the acyltransferase was interfered with when a large amount of lecithin was present in the dispersion. In addition, the amount of SDS required for maximum cholesterol esterification was not affected by the amount of lecithin present in the dispersion. These results suggest that the action of cholesterol sulfate on the acyltransferase is different from that of SDS.
Human plasma contains lecithin-cholesterol acyltransferase [EC 2. 3.1.43] (LCAT), which catalyzes the transfer of an acyl group from the /3-position of lecithin to cholesterol (7). The acyltransferase is inhibited by cholesterol ester and lysolecithin, which are the endproducts of this reaction {2, 3). On the other hand, it has been reported that cholesterol is converted to cholesterol sulfate by the transfer of a sulfonyl group from ascorbic 2-sulfate to cholesterol in mammalian Vol. 80, No. 4, 1976
tissues (4). Recently, it has been found that cholesterol sulfate is a normal constituent of human erythrocytes and plasma and has a protective action against hypotonic hemolysis of human blood cells as an amphipathic lipid (5). However, its biological function is not clear, In this paper, the effects of cholesterol sulfate and SDS, a well-known surfactant, on LCAT in human plasma were compared,
729
730
M. NAKAGAWA and S. KOJIMA
tubes, flushed with N t , sealed and incubated at 37° for 3 hr with constant shaking. After MATERIALS AND METHODS incubation, extraction and separation of lipids Preparation of the Enzyme—As an enzyme and measurement of the radioactivity were source for LCAT, human plasma was obtaine'd conducted as described previously (6~). The] from outdated human blood containing 0.15 determipation of free cholesterol in human volume of anticoagulant solution (citric acid, plasma 'was performed by the procedure desodium citrate, and glucose) by centrifigation scribed by Muesing and Nishida (9). The and was then dialyzed against Tris-HCl buffer, protein content was determined by the procedure described by Lowry et al. (10) with pH 7.0, ionic strength 0.1. Compounds—Cholesterol and SDS were ob- crystalline bovine serum albumin as a standard. tained from Kanto Chemical Co. (Tokyo, Japan). [3H]Cholesterol was purchased from RESULTS AND DISCUSSION New England Nuclear Corp. (Boston, Mass., U.S.A.) and purified as described previously We have previously reported that when soni(6). Bovine serum albumin was purchased cated dispersions of lecithin and cholesterol from Sigma Chemical Co. (St. Louis, Mo., mixtures are used as substrates for LCAT in U.S.A.). Lecithin was prepared from egg yolk human plasma, cholesterol esterification inby the method of Faure (7) and purified by creases linearly with time up to 3 hr (11). Therefore, using similar experimental condisilicic acid column chromatography. Thintions, the effect of cholesterol sulfate on the layer chromatography of the purified lecithin acyltransferase was compared with that of on silica gel G plates with chloroform-methSDS, which is a well-known amphipathic and anol-water (65 : 25 : 4, v/v) as a developing surface-active compound. As shown in Fig. 1, solvent gave a single spot of lecithin. Potassium cholesterol sulfate was synthesized chemically and purified by the procedure of Mumma (8). Preparation of Substrate Dispersion—Sonicated dispersions of lecithin and cholesterol mixtures were prepared as described previously (6). We confirmed by thin-layer chromatography that no degradation of lecithin to lysolecithin occurred during sonication. The lecithin/cholesterol molar ratios of the dispersions used were 2.2, 5.4, and 6.2. The radioactivity and amount of free cholesterol in dispersions having molar ratios of 5.4 and 6.2, and 2.2 were 0.2 ^Ci/0.16 /imole/ml incubation medium 0 10" 5 10~4 10" 1 CHOLESTEROL SULFATE OR SDS (M) and 0.2 ^Ci/0.13 /^mole/ml incubation medium, respectively. The determination of cholesterol Fig. 1. Effect of cholesterol sulfate (curves 3 and 4) and phospholipid phosphorus contents was per- and SDS (curves 1 and 2) on cholesterol esterification in the presence (curves 2 and 4) or absencs (curves formed as described previously (fJ). Enzyme Assay — The incubation mixture 1 and 3) of bovine serum albumin (30 mg/ml). The incubation medium contained 0.1 ml of the dispersion contained 0.1 ml of the dispersion and 0.2 ml (lecithin/cholesterol molar ratio of 6.2), 0.2 ml of of human plasma. The final volume was ad- human plasma and various amounts of cholesterol justed to 0.5 ml with Tris-HCl buffer, pH 7.0, sulfate or SDS. The final volume was adjusted to ionic strength 0.1. Human plasma used for 0.5 ml with Tiis-HCl buffer and the mixture was the incubation was diluted with Tris-HCl buffer incubated at 37° for 3 hr. The percentage of radioto give a protein content of 60 mg per ml. active cholesterol esterified during incubation is given The samples were placed in 15 ml screw-capped on the ordinate. / . Biochem.
AMPHIPATHIC MOLECULES AFFECTING LCAT
the'acyltransferase activity was" depressed at concentrations of cholesterol sulfate • higher than 1 X 1 0 ~ 4 M (curve 3) and decreased to approximately 79%, 27%, and 23% of the control level on the addition of lxlO~ 4 M, 5xlO~ 4 M, and 1X 10~8 M (467 fig per ml of incubation medium) cholesterol sulfate, respectively. The inhibitory effect of cholesterol sulfate reached a plateau at a concentration of approximately l x l O - ' M cholesterol sulfate. The concentration of cholesterol sulfate required for 50% inhibition of the acyltransferase was approximately 3 X 10~4 M (140 fig per ml of incubation medium). Since Bleau et al. (5) have reported that approximately 360 fig of cholesterol sulfate is contained per 100 ml of human plasma as a normal constituent, the cholesterol sulfate concentration required for 50% inhibition was approximately 40 times greater than the concentration in human plasma. However, assuming that the cholesterol in human plasma reached equilibrium with the radioactive cholesterol in the substrate dispersion during incubation, the cholesterol sulfate concentration required for 50% inhibition corresponds to approximately two-thirds of the amount of free cholesterol (approximately 221 fig per ml of incubation medium) in the incubation medium with the dispersion and human plasma which contained approximately 390 pg cholesterol per ml of plasma. Therefore, although the mechanism of inhibitory action of cholesterol sulfate on the acyltransferase is not clear, the interaction of cholesterol sulfate with the enzyme or high density lipoprotein (1.125
