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1.
Inhibition of lecithin-cholesterol acyltransferase by diphytanoyl phosphatidylcholine 总被引:2,自引:0,他引:2
H J Pownall Q Pao H L Brockman J B Massey 《The Journal of biological chemistry》1987,262(19):9033-9036
Model high density lipoproteins containing human apolipoprotein A-I, cholesterol, and a variety of phosphatidylcholines (PCs) have been prepared and tested. The PCs included 1-palmitoyl-2-oleoyl PC (POPC) and its diether analog 1-O-hexadecyl-2-oleyl PC (POPC ether), 1,2-diphytanoyl PC (DPhPC), 1-palmitoyl-2-phytanoyl PC, and 1-phytanoyl-2-palmitoyl PC. All ester PCs were good acyl donors for the transesterification of cholesterol catalyzed by human lecithin-cholesterol acyltransferase except DPhPC, which showed no reactivity. The PCs containing one phytanoyl chain donated an acyl chain to cholesterol as fast as non-branched fatty acyl chains. However, the competitive inhibition of lecithin-cholesterol acyltransferase by POPC ether and DPhPC was similar, and both lipids formed a macromolecular matrix that supported the reactivity of other ester PC substrates. The bulk of physicochemical properties of model high density lipoproteins composed of DPhPC were indistinguishable from those of POPC ether. These properties included 1) alpha-helical content of the apoprotein as assessed by circular dichroism, 2) microviscosity as determined from the fluorescence polarization and lifetime of the probe 1,6-diphenyl-1,3,5-hexatriene, 3) macromolecular weight based upon analytical gel filtration chromatography, and 4) surface polarity revealed by the fluorescence of 6-propionyl-2(dimethylamino)naphthalene. The only major difference in a physicochemical property was that the molecular surface area of DPhPC (area = 69 A2 at collapse pressure) determined by monolayer methods was 17 A2 greater than that of POPC (area = 53 A2 at collapse pressure) at all surface pressures measured. We suggest that the properties of DPhPC in being enzymatically nonreactive but a competitive inhibitor are due to its much larger size and that the active site of lecithin-cholesterol acyltransferase cannot bind phospholipid substrates in a catalytically productive way if they have surface areas of 70 A2 or more. 相似文献
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Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma 总被引:1,自引:0,他引:1
Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT. 相似文献
4.
The effect of the inclusion of phosphatidylethanolamine (PE), a phospholipid with unusual packing properties, on the substrate properties of protein-lipid complexes toward lecithin-cholesterol acyltransferase (LCAT) has been studied. Recombinant particles of apolipoprotein A-I with dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and cholesterol were prepared at a molar ratio of 1:140:14 (A-I/DMPC/cholesterol) or 1:70:70:14 (A-I/DMPC/DLPE/cholesterol); the efficiency of cholesterol incorporation into complexes containing phosphatidylethanolamine was found to be very pH-dependent, with enhanced cholesterol incorporation at elevated pH values. By incubating the complexes with either purified human LCAT or the d greater than 1.21 g/ml fraction of rat serum as a source of LCAT activity, it was found that a high degree of cholesterol esterification could be achieved with either complex; however, the DLPE-containing complex possessed a much smaller Stokes' diameter than the DMPC-only particle despite compositional similarities between these complexes. With respect to particle diameter the DLPE-containing particles behaved more like complexes prepared with egg yolk lecithin than did complexes prepared with DMPC alone. When human LDL was added to the incubations to provide a source of additional cholesterol, the products were markedly different. Concomitant with an increased cholesteryl ester core was an increase in the protein stoichiometry in both types of particles, from 2 to 3 or 4 apo A-I per particle. The proportion of DLPE to DMPC in the products was reduced from 1:1 to 0.3:1, reflecting a preferential hydrolysis of PE by LCAT, and the Stokes' diameters of the DMPC-only and the DLPE-containing complexes were closely similar. We conclude that the presence of elevated proportions of certain phospholipid species may significantly alter both the physical properties of the particles and their substrate properties with regard to reactions with enzymes of lipid metabolism. 相似文献
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The activity of serum lecithin-cholesterol acyltransferase was increased on administration of phenobarbital to the rat. This effect was dependent on dose and elapsed time after administration of the drug. Phenobarbital did not stimulate lecithin-cholesterol acyltransferase activity when added to serum from normal animals in vitro. Presumably, phenobarbital increased serum lecithin-cholesterol acyltransferase activity by induction of the microsomal enzyme and subsequent secretion by the liver. 相似文献
7.
The metabolism of cholesterol derived from [3H]cholesterol-labeled low-density lipoprotein (LDL) was determined in human blood plasma. LDL-derived free cholesterol first appeared in large alpha-migrating HDL (HDL2) and was then transferred to small alpha-HDL (HDL3) for esterification. The major part of such esters was retained within HDL of increasing size in the course of lecithin-cholesterol acyltransferase (LCAT) activity; the balance was recovered in LDL. Transfer of preformed cholesteryl esters within HDL contributed little to the labeled cholesteryl ester accumulating in HDL2. When cholesterol for esterification was derived instead from cell membranes, a significantly smaller proportion of this cholesteryl ester was subsequently recovered in LDL. These data suggest compartmentation of cholesteryl esters within plasma that have been formed from cell membrane or LDL free cholesterol, and the role for HDL2 as a relatively unreactive sink for LCAT-derived cholesteryl esters. 相似文献
8.
The interaction of rat plasma lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles and with rat apo-A-I was studied in comparison with that of human plasma lecithin-cholesterol acyltransferase to clarify the reaction mechanism of rat plasma lecithin-cholesterol acyltransferase. The interaction of both human and rat lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles was investigated by gel permeation chromatography on Superose 12. Both enzymes had almost the same affinity to the vesicles. The affinity of rat enzyme to rat apo-A-I was stronger than that of human enzyme to human apo-A-I when estimated on the apo-A-I-Sepharose 4B column. When human apo-A-I was added to the human enzyme/vesicle mixture which contained the enzyme-vesicle complex, the enzyme was effectively dissociated from the complex. But when rat apo-A-I was added to the rat enzyme/vesicle mixture, apo-A-I-enzyme-vesicle complex was still recognized by its elution pattern on gel permeation chromatography. This suggests that the mixture of rat enzyme, rat apo-A-I, and vesicles, which are the major components in the rat lecithin-cholesterol acyltransferase reaction, forms a stronger complex than do the components of the human reaction. 相似文献
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Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme. 相似文献
12.
E L Gong A V Nichols K H Weisgraber T M Forte V G Shore P J Blanche 《Biochimica et biophysica acta》1989,1006(3):317-328
The primary objectives of this study were to determine whether analogs to native discoidal apolipoprotein (apo)E-containing high-density lipoproteins (HDL) could be prepared in vitro, and if so, whether their conversion by lecithin-cholesterol acyltransferase (LCAT; EC 2.3.1.43) produced particles with properties comparable to those of core-containing, spherical, apoE-containing HDL in human plasma. Complexes composed of apoE and POPC, without and with incorporated unesterified cholesterol, were prepared by the cholate-dialysis technique. Gradient gel electrophoresis showed that these preparations contain discrete species both within (14-40 nm) and outside (10.8-14 nm) the size range of discoidal apoE-containing HDL reported in LCAT deficiency. The isolated complexes were discoidal particles whose size directly correlated with their POPC:apoE molar ratio: increasing this ratio resulted in an increase in larger complexes and a reduction in smaller ones. At all POPC:apoE molar ratios, size profiles included a major peak corresponding to a discoidal complex 14.4 nm long. Preparations with POPC:apoE molar ratios greater than 150:1 contained two distinct groups of complexes, also in the size range of discoidal apoE-containing HDL from patients with LCAT deficiency. Incorporation of unesterified cholesterol into preparations (molar ratio of 0.5:1, unesterified cholesterol:POPC) resulted in component profiles exhibiting a major peak corresponding to a discoidal complex 10.9 nm long. An increase of unesterified cholesterol and POPC (at the 0.5:1 molar ratio) in the initial mixture, increased the proportion of larger complexes in the profile. Incubation of isolated POPC-apoE discoidal complexes (mean sizes, 14.4 and 23.9 nm) with purified LCAT and a source of unesterified cholesterol converted the complexes to spherical, cholesteryl ester-containing products with mean diameters of 11.1 nm and 14.0 nm, corresponding to apoE-containing HDL found in normal plasma. Conversion of smaller cholesterol-containing discoidal complexes (mean size, 10.9 nm) under identical conditions resulted in spherical products 11.3, 13.3, and 14.7 nm across. The mean sizes of these conversion products compared favorably with those (mean diameter, 12.3 nm) of apoE-containing HDL of human plasma. This conversion of cholesterol-containing complexes is accompanied by a shift of some apoE to the LDL particle size interval. Our study indicates that apoE-containing complexes formed by the cholate-dialysis method include species similar to discoidal apoE-containing HDL and that incubation with LCAT converts most of them to spherical core-containing particles in the size range of plasma apoE-containing HDL. Plasma HDL particles containing apoE may arise in part from direct conversion of discoidal apoE-containing HDL by LCAT. 相似文献
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Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface. 相似文献
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Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified from hog plasma by a highly efficient procedure. The final enzyme preparation was purified 30,000-fold over the starting material and was homogeneous as indicated by polyacrylamide gel electrophoreses in the presence of both SDS and urea. The purified hog lecithin-cholesterol acyltransferase had an apparent molecular weight of 66 000 on SDS-polyacrylamide gel electrophoresis and HPLC and was found to contain about 21.4% (w/w) carbohydrate-hexose, 11.3%; hexosamine, 1.9%; sialic acid, 8.2%. The amino acid composition analysis showed that hog lecithin-cholesterol acyltransferase contains four half cystines per mol; two cysteines were titrated at neutral pH with 5,5'-dithiobis(2-nitrobenzoic acid). Nearly all the phenolic groups were unavailable to the solvent at neutral pH, while they become exposed at around pH 11. Hog lecithin-cholesterol acyltransferase was found to be associated with HDL in the plasma and it prefers HDL as a substrate. The physicochemical properties of hog lecithin-cholesterol acyltransferase were generally similar to those of the human and the rat enzyme. 相似文献
16.
Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism 总被引:6,自引:0,他引:6
Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement of the phospholipase A2 and transacylase activities of the enzyme using proteoliposome substrates and following selective chemical modification of serine, histidine, and cysteine residues of pure homogeneous LCAT indicated the following catalytic mechanism: HS-Cys-E-Ser-OH + lecithin in equilibrium HS-Cys-E-Ser-O-FA + lysolecithin, HS-Cys-E-Ser-O-FA in equilibrium FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH + cholesterol-OH in equilibrium HS-Cys-E-Ser-OH + cholesterol-O-FA, where FA denotes fatty acid. Modification of 2 LCAT cysteine residues with 5,5'-dithiobis-(2-nitrobenzoic acid) or treatment with ferricyanide inactivated the transacylase but not the phospholipase A2 activity. Modification of 1 serine residue with phenylmethanesulfonyl fluoride or 1 histidine residue with diethyl pyrocarbonate inhibited cholesteryl ester formation and phospholipase A2 activity. Proteoliposome substrates protected both activities against chemical inactivation. Lecithin alone protected the phospholipase A2 activity against phenylmethanesulfonyl fluoride inactivation but not the transacylase against 5,5'-dithiobis-(2-nitrobenzoic acid) inactivation. Incubation of native LCAT with arachidonyl-CoA or the lecithin-apo-A-I proteoliposome resulted in acylation of three enzyme sites, only one of which was stable to neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- and thioesters were demonstrable in both cases. No transfer of arachidonic acid from iodoacetamide-modified LCAT to cholesterol occurred, indicating that the fatty-acylated serine residue cannot directly esterify cholesterol. Cholesterol arachidonate was formed upon incubation of phenylmethanesulfonyl fluoride-modified LCAT with arachidonyl-CoA. 相似文献
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E L Gong A V Nichols T M Forte P J Blanche V G Shore 《Biochimica et biophysica acta》1988,961(1):73-85
Using a cholate-dialysis recombination procedure, complexes of apolipoprotein A-I and synthetic phosphatidylcholine (1-palmitoyl-2-oleoylphosphatidylcholine (POPC) or dioleoylphosphatidylcholine (DOPC] were prepared in mixtures at a relatively high molar ratio of 150:1 phosphatidylcholine/apolipoprotein A-I. Particle size distribution analysis by gradient gel electrophoresis of the recombinant mixtures indicated the presence of a series of discrete complexes that included species migrating at RF values observed for discoidal particles in nascent high-density lipoproteins (HDL) in plasma of lecithin-cholesterol acyltransferase-deficient subjects. One of these complex species, designated complex class 6, formed with either phosphatidylcholine, was isolated by gel filtration and characterized at follows: discoidal shape (mean diameter 20.8 nm (POPC) and 19.0 nm (DOPC]; molar ratio, phosphatidylcholine/apolipoprotein A-I, 155:1 (POPC) and 130:1 (DOPC); and both containing 4 molecules of apolipoprotein A-I per particle. Incubation of class 6 complexes with lecithin-cholesterol acyltransferase (EC 2.3.1.43) and a source of unesterified cholesterol (low-density lipoprotein (LDL] was shown by electron microscopy to result in a progressive transformation of the discoidal particles (0 h) to deformable (2.5 h) and to spherical particles (24 h). The spherical particles (diameter 13.6 nm (POPC) and 12.5 nm (DOPC) exhibit sizes at the upper boundary of the interval defining the human plasma (HDL2b)gge (12.9-9.8 nm). The spherical particles contain a cholesteryl ester core that reaches a limiting molar ratio of approx. 50-55:1 cholesteryl ester/apolipoprotein A-I. The deformable particles assume a rectangular shape under negative staining and, relative to the 24-h spherical product, are enriched in phosphatidylcholine. Chemical crosslinking (by dimethyl suberimidate) of the isolated transformation products shows the 24-h spherical particle to contain predominantly 4 apolipoprotein A-I molecules; products produced after intermediate periods of time appear to contain species with 3 and 4 apolipoproteins per particle. Our in vitro studies indicate a potential pathway in the origins of large, apolipoprotein A-I-containing plasma HDL particles. The deformable species observed during transformation were similar in size and shape to particles observed in interstitial fluid. 相似文献
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Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2 总被引:1,自引:0,他引:1
C H Chen T H Forte B E Cahoon R N Thrift J J Albers 《Biochimica et biophysica acta》1986,877(3):433-439
The human liver cell line HepG2 was investigated for its synthesis and secretion of lecithin-cholesterol acyltransferase. The cells were grown to confluency in Eagle's minimal essential medium plus 10% fetal bovine serum. At the onset of the study, fetal bovine serum was removed and cells were grown in minimal essential medium only. At 6, 12, 24, and 48 h the cells were harvested, and the culture medium collected at each time point was assayed for lecithin-cholesterol acyltransferase mass and activity, cholesterol esterification rate, and apolipoprotein A-I mass. The rate of the enzyme secretion measured by both mass and activity was linear over 24 h of culture. The enzyme mass by radioimmunoassay was 1.7, 4.1, 7.9 and 13.7 ng/ml culture medium (or 8.3, 19.9, 38.5 and 66.7 ng/mg cell protein), respectively, and enzyme activity using an exogenous source of phosphatidylcholine/cholesterol liposomes containing apolipoprotein A-I as substrate was 85, 170, 315, and 402 pmol cholesterol esterified/h per ml culture medium (or 414, 828, 1534 and 1957 pmol cholesterol esterified/h per mg cell protein) for 6, 12, 24, and 48 h of culture, respectively. The endogenous cholesterol esterification rate of the culture medium was 47, 104, 224 and 330 pmol/h per ml and apolipoprotein A-I mass was 305, 720, 2400 and 3940 ng/ml culture medium over the same time frame. In contrast to culture medium, low levels of enzyme activity (approximately 10% of that in culture medium at 24 and 48 h) were observed in the extracts of HepG2 cells. The enzyme secreted by HepG2 was found to be similarly activated by apolipoprotein A-I, apolipoprotein E, or apolipoprotein A-IV, and was similarly inhibited by phenylmethylsulfonyl fluoride, dithiobisnitrobenzoate, p-hydroxymercuribenzoate, or iodoacetate as compared to human plasma enzyme. High-performance gel filtration of the culture medium revealed that the HepG2-secreted enzyme was associated with a fraction having a mean apparent molecular weight of approximately 200,000. We concluded that human hepatoma HepG2 cells synthesize and secrete lecithin-cholesterol acyltransferase, which is functionally homologous to the human plasma enzyme. 相似文献
19.
Carlos Soler-Argilaga Robert L. Russell Murray Heimberg 《Archives of biochemistry and biophysics》1977,178(1):135-139
The effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase was measured in rat serum following addition of Triton to the serum in vitro or after intravenous injection of the detergent. The inhibitory effect of Triton WR-1339 on activity of lecithin-cholesterol acyltransferase when the detergent was added in vitro was dose dependent and appeared to result from a direct action on the enzyme rather than from a physical modification of the substrate by the detergent. The serum half-life () of Triton WR-1339 injected intravenously in the rat was 23.1 ± 1.0 h. The inhibitory effect of Triton on serum LCAT activity when the detergent was given intravenously was also dose dependent and was reversed when the serum concentration of Triton decreased; under specific conditions, LCAT activity reached values higher than control. This behavior after treatment of the animal may be explained by increased concentration of the enzyme in the plasma, by stimulation of LCAT activity by the very low density lipoprotein or metabolites accumulating in the plasma of rats treated with Triton WR-1339, or by a combination of these factors. 相似文献
20.
N Dousset L Douste-Blazy 《Comptes rendus des séances de la Société de biologie et de ses filiales》1975,169(2):440-443
Plasma cholesterol and lecithin-cholesterol-acyltransferase activity are studied in irradiated rats. Ionizing radiations cause an increase of cholesterol levels in plasma, concerning mainly ester fraction. Lecithin-cholesterol-acyltransferase activity in plasma of irradiated rats is diminued 48 hours after exposure. This decreased rate of LCAT is probably the consequence of the post-irradiation hypercholesterolemia. 相似文献