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At least 1,153 records · Page 64Linked to original sources
PubMed1999

Effect of lecithinized superoxide dismutase (PC-SOD) on experimental pulmonary metastasis in mice.

The inhibitory effect of lecithinized superoxide dismutase (PC-SOD) on pulmonary metastasis in mice was investigated. In an experimental pulmonary metastasis model employing Meth A-T cells, significant and dose-dependent inhibition was observed after i.v. pre-administration of PC-SOD. Unmodified SOD (U-SOD) was also effective, but a 10-times higher dose was necessary to be significant. The pulmonary accumulation of Meth A-T cells labeled with 5-[125I]iodo-2'-deoxyuridine was not reduced by either PC-SOD or U-SOD, and neither of the compounds decreased pulmonary MPO activity. However, PC-SOD increased pulmonary SOD activity for longer, compared with U-SOD. In vitro addition of PC-SOD dose-dependently suppressed the growth of Meth A-T cells, while U-SOD had little effect. The combination of PC-SOD and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitric oxide (NO)-generating agent, had an additive effect. It was also found that PC-SOD prevented a decrease of pulmonary NOx level following tumor cell inoculation. It was concluded that PC-SOD possessed antimetastatic activity, and its potency was superior to that of U-SOD. These results suggest that PC-SOD may prevent the excessive formation of oxygen radicals and peroxynitrite (ONOO-) which cause cell damage and facilitate tumor metastasis.

Animals↗
PubMed2001

Acyl-CoA: retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activation during the lipocyte phenotype induction in hepatic stellate cells.

We have examined retinol esterification in the established GRX cell line, representative of hepatic stellate cells, and in primary cultures of ex vivo purified murine hepatic stellate cells. The metabolism of [3H]retinol was compared in cells expressing the myofibroblast or the lipocyte phenotype, under the physiological retinol concentrations. Retinyl esters were the major metabolites, whose production was dependent upon both acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT). Lipocytes had a significantly higher esterification capacity than myofibroblasts. In order to distinguish the intrinsic enzyme activity from modulation of retinol uptake and CRBP-retinol content of the cytosol in the studied cells, we monitored enzyme kinetics in the purified microsomal fraction. We found that both LRAT and ARAT activities were induced during the conversion of myofibroblasts to lipocytes. LRAT induction was dependent upon retinoic acid, while that of ARAT was dependent upon the overall induction of the fat storing phenotype. The fatty acid composition of retinyl-esters suggested a preferential inclusion of exogenous fatty acids into retinyl esters. We conclude that both LRAT and ARAT participate in retinol esterification in hepatic stellate cells: LRAT's activity correlates with the vitamin A status, while ARAT depends upon the availability of fatty acyl-CoA and the overall lipid metabolism in hepatic stellate cells.

Journal Article↗
PubMed2000

Headgroup specificity of lecithin cholesterol acyltransferase for monomeric and vesicular phospholipids.

In this study, we investigated how the nature of the phospholipid head group and the macromolecular structure of the phospholipid, either as a monomer or incorporated into a lipid matrix, influence the activity of lecithin cholesterol acyltransferase (LCAT). As substrates we used 1,2-bis-(1-pyrenebutanoyl)-phosphatidylcholine, 1, 2-bis-(1-pyrenebutanoyl)-phosphatidylethanolamine and 1, 2-bis-(1-pyrenebutanoyl)-phosphatidyl-alcohols, either as monomers or incorporated into small unilamellar vesicles consisting of dipalmitoylphosphatidylcholine ether. The rate of hydrolysis of the pyrene-labeled phospholipids was determined both by fluorescence and by high performance liquid chromatography. V(max) and K(m) were calculated for the different substrates. The data show that V(max) is 10- to 30-fold higher for the hydrolysis of monomeric phosphatidylcholine (PC) compared to phosphatidylethanolamine (PE) and the phosphatidylalcohols, while K(m) values are comparable. When the fluorescent substrates were incorporated into dipalmitoylphosphatidylcholine ether vesicles, we observed a 4- to 10-fold increase of V(max) for PE and the phosphatidylalcohols, and no significant change for K(m). V(max) for PC remained the same. Natural LCAT mutants causing Fish-Eye Disease (FED) and analogues of these mutants expressed in Cos-1 cells, had similar activity on monomeric PC and PE. These data suggest that the activity of LCAT is determined both by the molecular structure of the phospholipid and by its macromolecular properties. The LCAT activity on monomeric substrates decreases as: phosphatidylcholine&z. Gt;phosphatidylethanolamine congruent withphosphatidylpropanol congruent withphosphatidylethanol congruent withphosphatidylethyleneglycol. The incorporation of PE and the phosphatidylalcohols into a matrix of dipalmitoylphosphatidylcholine decreases the specificity of the phospholipid head group.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Importance of the free sulfhydryl groups of lecithin-cholesterol acyltransferase for its sensitivity to oxidative inactivation.

Lecithin-cholesterol acyltransferase (LCAT) of human plasma is known to be highly susceptible to oxidative inactivation, although the mechanism of this inactivation is unknown. We tested the hypothesis that the high sensitivity of the enzyme is due to the derivatization of its two free SH groups flanking the active site pocket. Modification of the SH groups with a reversible inhibitor protected the enzyme against oxidative inactivation. Mutagenesis of either of the cysteines to glycine increased the resistance of the enzyme, which retained 46% of activity in presence of 150 microM Cu(2+), compared to only 27% of the activity retained by the wild type enzyme (WT). Replacement of both the cysteines with glycines resulted in retention of over 65% activity. Cysteine replacement similarly protected the enzyme from inactivation by the oxidized substrate. Chicken LCAT, which has only one cysteine (Cys(26)), was more resistant than the human enzyme. Introduction of an additional cysteine corresponding to the second cysteine in human LCAT (N184C) resulted in increased susceptibility of chicken enzyme (87% loss of activity in presence of 150 microM Cu(2+), compared to 55% loss in WT). Substitution of the lone cysteine with glycine (C26G) resulted in a more resistant enzyme, which lost <40% activity under the same conditions. These results show that the primary targets of the oxidizing agents or the products of oxidation are the SH groups of the enzyme, whose derivatization leads to steric inhibition of the activity.

Animals↗
PubMed2003

Natural mutations of apolipoprotein A-I impairing activation of lecithin:cholesterol acyltransferase.

Five natural mutations of apolipoprotein A-I (apoA-I), apoA-I(A95D), apoA-I(Y100H), apoA-I(E110K), apoA-I(V156E) and apoA-I(H162Q), were studied for their ability to activate lecithin:cholesterol acyltransferase (LCAT). Mutants apoA-I(E110K), apoA-I(V156E) and apoA-I(H162Q) had an impaired ability to activate LCAT. Combined with data on other apoA-I mutants this finding is consistent with the idea that the central region between amino acids 110 and 160 is likely to be the "active site" of apoA-I involved in the interaction with LCAT and that a specific sequence of apoA-I is required for activation of the enzyme.

Amino Acid Sequence↗
PubMed1999

Role of lecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: studies with platelet-activating factor-acetyl hydrolase-deficient plasma.

To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-14C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2000

Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source.

Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.

Animals↗
PubMed2003

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins.

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.

Amino Acids↗
PubMed1980

Lecithin: cholesterol acyltransferase activity and fatty acid composition of erythrocyte phospholipids in Friedreich's ataxia.

In a study of the fatty acid composition of erythrocyte membrane phospholipids in Friedreich's ataxia, a lower percentage of linoleic acid in phosphatidylcholine was demonstrated. An enzyme involving the exchange of lipids between plasma and erythrocyte membrane, lecithin: cholesteryl acyltransferase (LCAT) was also studied. It was found that the LCAT activity had a trend towards low values. However, crossing-over studies indicated that when the LCAT enzyme of patients was exposed to its own substrate it gave low activity values but that the result reverted to normal when control substrate was used.

Acyltransferases↗
PubMed1982

Oral lecithin and linoleic acid in Friedreich's ataxia: I. Design of the study, material and methods.

A clinical and biochemical evaluation of twenty-two patients with Friedreich's Ataxia and ten normal controls was undertaken in 1980 to assess the effect of lecithin and linoleic acid supplements on the course of the disease. The trial consisted of two consecutive six months periods on either supplements in a double-blind crossover fashion. Clinical appraisal was performed with regards to the following parameters: joints mobility, muscle strength, equilibrium, coordination, motor accuracy, speech and numerous day to day activities. Blood samples were obtained at the beginning and in the course of the trial for enzymatic determinations. This paper describes the methodology of the study.

Administration, Oral↗
PubMed1995

Lecithin:cholesterol acyltransferase reaction on cellular lipid released by free apolipoprotein-mediated efflux.

Lecithin:cholesterol acyltransferase (LCAT) reaction was studied in free apolipoprotein-mediated cellular lipid efflux from mouse peritoneal macrophages and human skin fibroblasts. When the cells were incubated with lipid-free human apolipoproteins (apo) A-I or A-II, pre-beta high density lipoprotein (HDL) particles were generated by removing cellular cholesterol and phospholipid. Cholesterol was esterified by LCAT in such particles generated with human apoA-I, but not in those with apoA-II. The reactivity of the apoA-I-pre-beta-HDL particles with LCAT was in the same order as that in human plasma HDL and in phosphatidylcholine/cholesterol unilamellar vesicles activated by apoA-I when compared on the rate of percent cholesterol esterification. However, cholesterol efflux mediated by apoA-I was not enhanced by active cholesterol esterification in the medium from either type of cells. Thus, it is unlikely the LCAT reaction on newly generated pre-beta-HDL directly causes further cellular cholesterol efflux. In control experiments, LCAT esterified cholesterol on human plasma HDL in the cell medium regardless of its origin, either HDL or cells. Cholesterol esterification on HDL was unable to enhance cellular cholesterol efflux significantly but reduced the influx of cholesterol from HDL to cell, resulting in the increase of net efflux of cellular cholesterol, in agreement with the results previously demonstrated.

Animals↗
PubMed1995

Comparison of lipid-binding and lecithin:cholesterol acyltransferase activation of the amino- and carboxyl-terminal domains of human apolipoprotein E3.

To extend the characterization of the functional domains of apolipoprotein E (apoE), the amino-(residues 1-191, 22-kDa) and carboxyl-terminal (residues 216-299, 10-kDa) fragments were tested for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. A disulfide bond linking helices 2 and 3 of the four-helix bundle amino-terminal domain was introduced by mutating threonine-57 to cysteine (Thr57-->Cys) in apoE3 (cysteine at position 112) to determine the influence of the disulfide bond on the properties of this domain. Lipid-binding properties were determined by the ability to form complexes with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine, assessed by measuring decreases in turbidity as a function of temperature. The results demonstrate that the relative lipid binding efficiencies were intact apoE3 approximately 10-kDa fragment > 22-kDa fragment > Thr57-->Cys variant. In addition, free, non-lipid-associated protein was observed with the two 22-kDa fragments but not with intact apoE3 or the 10-kDa fragment. The transition temperatures determined by fluorescence polarization were higher for the DMPC complexes with intact apoE3 and with 22- and 10-kDa fragments (25.5 degrees C) than with the 22-kDa Thr57-->Cys variant (23.5 degrees C), suggesting that the variant fragment possessed the lowest affinity for lipid. Attenuated total reflection infrared measurements of the complexes indicated that the long axes of the alpha-helices of the various apoE forms were parallel to the acyl chains of the phospholipid bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein E3↗
PubMed1993

Roles of cysteines in human lecithin:cholesterol acyltransferase.

Human lecithin:cholesterol acyltransferase (LCAT, E.C.2.3.1.43) is a serine-type esterase that contains six cysteines, two of which, Cys31 and Cys184, are free. The remaining cysteines form disulfide links. One of these is between Cys50 and Cys74 and the other is between Cys313 and Cys356. The cDNA of LCAT and mutants in which one or two of the six cysteines were replaced by glycine was expressed in COS-6 cells. Polymerase chain reactions and Northern blot analysis indicated that LCAT mRNA was produced by all transfectants. Western blots of all transfected cells probed with a polyclonal antibody revealed intracellular LCAT. Substitution of glycine for either Cys50, Cys74, Cys313, or Cys356 was associated with a nearly total absence of activity in the medium. No protein was secreted when glycine replaced either of the amino acid residues that link Cys313 and Cys356. The small amounts of the Cys50-->Gly and Cys74-->Gly mutants found in the medium had specific activities that were much lower than that of the wild-type LCAT. All other transfectants secreted immunologically measurable amounts of active enzyme. Mutants in which one or both free cysteines, Cys31 and Cys184, were replaced with glycine were less active than the wild type and only partially inhibited by a sulfhydryl blocking reagent. The substrate specificities of the Cys31-->Gly and Cys184-->Gly mutants differed from that of the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Effects of site-directed mutagenesis on the N-glycosylation sites of human lecithin:cholesterol acyltransferase.

There are four potential N-glycosylation site (Asn-X-Ser/Thr) in human lecithin:cholesterol acyltransferase (LCAT, residues 20, 84, 272, and 384). To study the role of the N-linked sugars, the codon for Asn at these positions was replaced with one for Thr (AAC to ACC). The wild-type and mutant LCAT cDNAs were used to transfect COS-6 cells from which RNA was isolated; cDNAs were synthesized by reverse transcription and subjected to the polymerase chain reaction, which showed that all transfectants synthesized LCAT-specific mRNA. No intracellular or secreted LCAT was detected with the Asn272-->Thr transfectants, indicating that this residue is essential for intracellular processing. All other single-point transfectants were secretion-competent. Although there was detectable LCAT protein inside the cells and in the media of the transfectant, Asn84-->Thr, its specific activity and secreted amount were only 26% and 58% of the wild type, respectively. This implies that Asn84 is critical for full activity but not for intracellular processing. The amount secreted, specific activity, and Vmax of LCAT (Asn20-->Thr) were similar to those of the wild-type LCAT. LCAT (Asn384-->Thr) differed from the wild-type LCAT only by a lower Km. These results suggest that glycosylation at residues 20 and 384 is not essential for intracellular processing, secretion, or activity.

Amino Acid Sequence↗
PubMed1991

Metabolism of low-density lipoprotein free cholesterol by human plasma lecithin-cholesterol acyltransferase.

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.

Carrier Proteins↗
PubMed1991

Structure-function relationships in human lecithin:cholesterol acyltransferase. Site-directed mutagenesis at serine residues 181 and 216.

The functions of serine residues at positions 181 and 216 of human plasma lecithin:cholesterol acyltransferase have been studied by site-directed mutagenesis. The serine residue at either site was replaced by alanine, glycine, or threonine in LCAT secreted from stably transfected CHO cells. All substitutions at position 181 gave rise to an enzyme product that was normally secreted but had no detectable catalytic activity. On the other hand, all substitutions at position 216 gave active products, whose activity was fully inhibitable by the serine esterase inhibitor diisopropyl fluorophosphate (DFP). A secondary (although not direct) role for serine-216 was indicated by a 14-fold increase in catalytic rate when this residue was substituted by alanine. Sequence comparison with other lipases suggests that serine-216 may be at or near the hinge of a helical flap displaced following substrate binding. These data strengthen the structural-functional relationship between LCAT and other lipases.

Amino Acid Sequence↗
PubMed1992

Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.

Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗
PubMed1992

Regulation of the concentration of pre beta high-density lipoprotein in normal plasma by cell membranes and lecithin-cholesterol acyltransferase activity.

A minor fraction of plasma high-density lipoprotein (pre beta-1 HDL) has been shown to promote cholesterol efflux from peripheral cell membranes [Castro, G. R., & Fielding, C. J. (1988) Biochemistry 27, 25-29]. When isolated native plasma is incubated at 37 degrees C, this fraction is specifically decreased. On the other hand, the level of plasma pre beta-1 HDL is fully protected in the presence of even very low levels of fibroblasts, vascular smooth muscle cells, or macrophages. Blood cells were completely inactive in maintaining plasma pre beta-1 HDL levels in the absence of peripheral cells, even at the relatively high levels present in whole blood. The loss of pre beta-1 observed in isolated plasma was dependent upon lecithin-cholesterol acyltransferase (LCAT) activity. These data suggest that reverse cholesterol transport catalyzed by pre beta-1 HDL, and subsequent LCAT-mediated cholesterol esterification, is directly dependent upon the interaction between this HDL species and competent peripheral cells.

Cell Membrane↗