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At least 1,009 records · Page 56Linked to original sources
PubMed1993

Reaction of lecithin: cholesterol acyltransferase with a water soluble substrate: effects of surfactants.

The reaction of fatty acid esters of p-nitrophenol, including the butyrate ester (PNPB) with lecithin: cholesterol acyltransferase (LCAT) has already been described (Bonelli, F.S. and Jonas, A. (1989) J. Biol. Chem. 264, 14723-14728) as a means to investigate the events at the active site of LCAT in the absence of interfacial interactions. Since various surfactants at low concentrations are known to affect the reaction of LCAT with particulate substrates, we set out to analyze their effects on the enzyme in solution using the PNPB substrate to monitor enzyme activity. All the surfactants studied by us (sodium dodecyl sulfate (SDS), dodecyltrimethyl ammonium bromide (DTAB), sodium laurate, sodium cholate, Triton X-100, and BIGCHAP) were able to interact with LCAT below their critical micellar concentrations. The ionic detergents caused inhibition of LCAT at concentrations ranging from 10(-4) to 10(-3) M, whereas the two nonionic detergents actually activated the enzyme in a similar concentration range. From the kinetic constants, the patterns of inhibition, and the well documented effects of the detergents on other proteins, we propose that SDS binds cooperatively to LCAT and elicits inhibitory structural changes; laurate and cholate bind to specific sites either in the active site cavity or in negative effector sites elsewhere; and the nonionic detergents may produce a slight interfacial activation of the phospholipase reaction near their critical micellar concentrations.

Binding Sites↗
PubMed1995

Role of N-linked glycosylation of lecithin:cholesterol acyltransferase in lipoprotein substrate specificity.

Lecithin:cholesterol acyltransferase (LCAT) is responsible for the formation of cholesteryl ester in plasma. LCAT is a glycoprotein which has a carbohydrate content estimated to be approx. 25% of its total mass. Previous studies of recombinant LCAT have characterized the function of the four N-linked glycosylation sites of LCAT with respect to reconstituted HDL analogue substrates. In order to investigate the relationship between N-linked glycosylation and the ability of LCAT to esterify cholesterol in native plasma lipoproteins, we have expressed a series of mutant LCAT cDNAs in which each of the four glycosylation consensus sequences was eliminated individually. All mutant LCAT proteins were secreted by stably transfected baby hamster kidney cells. The ability of mutant LCATs to esterify cholesterol in purified native lipoproteins indicated that the elimination of the carbohydrate chain at position 20 of recombinant LCAT was associated with a lower activity than the wild type enzyme when HDL was used as a substrate, but no inhibitory effect was observed when LDL was used as a substrate. A mutant enzyme with a substitution of Asn-84-->Gln or Asn-272-->Gln displayed a decreased ability to esterify cholesterol in either HDL or LDL. In contrast, the loss of a carbohydrate chain at position 384 was associated with an increase in enzyme activity for both HDL (1.5-fold) and LDL (2.5-fold) substrates. Kinetic analysis of these recombinant enzymes indicated that the apparent Km values for cholesterol in either HDL or LDL were not affected, but that the differences in activities were due to changes in the apparent Vmax. Heat inactivation studies were performed to assess the role of specific carbohydrate groups in enzyme stability. Loss of a carbohydrate chain at position 20, 272 or 384 decreased thermostability of LCAT whereas a mutation at position 84 did not affect thermostability. These results suggest that individual carbohydrate chains confer specific structural and functional properties to LCAT.

Animals↗
PubMed1995

Regulation of lecithin:cholesterol acyltransferase by TGF-beta and interleukin-6.

The human hepatoma derived HepG2 cells were treated with transforming growth factor-beta (TGF-beta) or interleukin-6 (IL-6) +/- dexamethasone. The effects of treatment on lecithin:cholesterol acyltransferase (LCAT) catalytic activity and mRNA level as well as on the apolipoprotein A-I (apo A-I) mRNA level were determined. Both the LCAT activity in medium from treated HepG2 cells and the LCAT mRNA level were decreased by TGF-beta. There was no significant effect of IL-6 +/- dexamethasone, neither on the LCAT activity nor on LCAT mRNA levels. Treatment with dexamethasone alone resulted in a decreased LCAT activity in spite of a slight increase in LCAT mRNA level. The apo A-I mRNA level was reduced after treatment with TGF-beta and increased after treatment with IL-6 +/- dexamethasone and dexamethasone alone. To analyze if the effects on mRNA levels were caused by transcriptional or post-transcriptional mechanisms, run-on experiments on isolated nuclei from treated HepG2 cells and mRNA degradation experiments were performed. The transcription rate of the LCAT gene was not affected by TGF-beta, but was increased (50-100%) after treatment with IL-6 +/- dexamethasone and dexamethasone alone. The transcription rate of the apo A-I gene was reduced (20%) by TGF-beta and increased (30-60%) by IL-6 +/- dexamethasone and dexamethasone alone. Both dexamethasone and TGF-beta increased the rate of LCAT mRNA degradation. These results show that the reduced LCAT mRNA level after treatment with TGF-beta was caused by post-transcriptional mechanisms.

Apolipoprotein A-I↗
PubMed1994

Interaction between apo A-I-containing lipoproteins and lecithin:cholesterol acyltransferase.

HDL2 and HDL3 subfractions of two species of apo A-I-containing lipoprotein, one containing only apo A-I (LpA-I) and the other containing both apo A-I and apo A-II (LpA-I/A-II), were tested for reactivity to lecithin:cholesterol acyltransferase (LCAT). These subfractions and their mixtures were incubated with lipoprotein-deficient plasma (LCAT source), and the rate of cholesterol esterification and kinetic parameters were determined. Apparent Vmax (appVmax) and apparent Km (appKm) for HDL2 subfractions of LpA-I and LpA-I/A-II were significantly lower than those of their HDL3 counterparts. Differences between subfractions were much more prominent in LpA-I than in LpA-I/A-II. appVmax of the HDL2 subfraction of LpA-I (LpA-IHDL2) was one-fifth, and appKm was one-third of those for the HDL3 subfraction (LpA-IHDL3). appVmax and appKm of LpA-IHDL2 were both lowest among the apo A-I-containing lipoprotein subfractions. When LpA-IHDL2 was added to other subfractions, the molar rate of cholesterol esterification was suppressed. Since LpA-IHDL2 consists of a particle 11.1 nm in diameter, our observations suggest that LpA-IHDL2 suppresses cholesterol esterification in apo A-I-containing lipoprotein, possibly by displacing LCAT from other subfractions with higher appKm and higher appVmax to 11.1 nm LpA-I particles with lower appKm and lower appVmax. All of these data suggest that the relative amount of 11.1 nm LpA-I particles in plasma regulates the reactivity of apo A-I-containing lipoprotein to LCAT and may play a key role on the production of cholesteryl esters in plasma.

Apolipoprotein A-I↗
PubMed1994

Substrate and positional specificities of human and mouse lecithin-cholesterol acyltransferases. Studies with wild type recombinant and chimeric enzymes expressed in vitro.

Human lecithin-cholesterol acyltransferase (LCAT) preferentially attacks sn-1 position of 16:0-20:4 phosphatidylcholine (PC), producing more 16:0 cholesteryl ester (CE) than 20:4 CE. In contrast, rat and mouse LCATs produce mostly 20:4 CE from the same PC. To understand the structural basis for this difference in positional specificity, we studied the specificities of recombinant mouse and human LCATs and several chimeric constructs of the two. The rLCATs retained the substrate and positional specificities of the plasma enzymes when expressed in COS-1 cells. Human and mouse LCAT cDNAs were each cleaved into three fragments, recombined in various combinations, and the chimeric products were analyzed for their specificities. When the N-terminal, or (and) C-terminal segments of human LCAT were replaced by the corresponding mouse LCAT segments, the chimeric products exhibited the specificity of intact human enzyme. However, when the middle segment, containing the residues 130-306 was replaced by the corresponding mouse LCAT segment, the enzyme exhibited the specificity of mouse LCAT. Similarly, the mouse rLCAT exhibited the specificity of human enzyme when its central segment, but not its N-terminal or C-terminal segment was replaced by the corresponding segment from human LCAT. These results show that the substrate and positional specificities of LCAT are controlled by the central domain of LCAT protein, corresponding to the amino acid residues 130-306.

Amino Acid Sequence↗
PubMed1994

The condensing effects of egg lecithin and cholesterol on triolein monolayers are inhibited by substitution of one saturated acyl chain in the triacylglycerol.

Previous work showed that the clearance from plasma of chylomicron-like emulsions injected intravenously was affected by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing triolein (OOO) as the only triacylglycerol, clearances were decreased by a single saturated chain in emulsions containing 1,3-dioleoyl-2-stearoyl-sn-glycerol (OSO), 1,2-dioleoyl-3-stearoyl-sn-glycerol (OOS) or 1-stearoyl-2,3-dioleoyl-sn-glycerol (SOO). The differences in clearance may reflect physical differences at the oil-water interface related to chain interactions of the triacylglycerol structures with other lipid components. In the present work lipid monomolecular films at the air-water interface were used to establish the capacity of OOO to interact with the pure synthetic triacylglycerols OOS and SOO, and the capacity of OOS and SOO to co-exist in monolayers of lecithin and of cholesterol was compared with OOO. Substituting one oleoyl chain by a stearoyl chain induced a 20% condensation in monomolecular films of the pure triacylglycerols. Mixtures of OOO with either pure egg yolk phosphatidylcholine or cholesterol also showed substantial condensing effects. In contrast substituting one oleoyl chain by a stearoyl chain substantially lessened the condensing effects. At surface pressures above the collapse pressure of the pure triacylglycerols, substantially more OOO than OOS or SOO was retained in mixed monolayers with phosphatidylcholine. These differences could underlie the effects on metabolism of saturated chains in emulsion triacylglycerols.

Chemical Phenomena↗
PubMed1995

Gas-phase cigarette smoke inhibits plasma lecithin-cholesterol acyltransferase activity by modification of the enzyme's free thiols.

Cigarette smoking is associated with an increased risk of premature atherosclerosis. The underlying mechanisms responsible for this association are unknown. Recent work from this laboratory has shown that ex vivo exposure to plasma to gas-phase cigarette smoke (CS) produces a rapid inhibition of lecithin-cholesterol acyltransferase (LCAT) activity and crosslinking of HDL-apolipoproteins. The goal of the present study was to investigate the mechanism(s) by which CS inhibited LCAT and modified HDL. When dialyzed human plasma (12 ml) was exposed to the gas-phase of an equivalent of 1/8 of a cigarette (one 'puff') at 15 min intervals for 3 h, LCAT activity was reduced by 76 +/- 1% compared to controls; supplementation of plasma with glutathione produced a dose-dependent protection of LCAT activity where at the highest concentration (1 mM) 78% protection was observed. A similar protection was obtained with N-acetyl cysteine (1 mM). In addition to LCAT inhibition, HDL-apolipoproteins were crosslinked after 3 h exposure of plasma to CS; crosslinking was reduced by the addition of either glutathione or N-acetyl cysteine to plasma. The amino compounds N-acetyl lysine, N-acetyl arginine, and aminoguanidine failed to protect LCAT and HDL indicating a specificity with regard to the ability of free thiols to buffer the deleterious components of CS which inhibited LCAT and crosslinked HDL-apolipoproteins. Since LCAT contains two free cysteine residues (Cys-31 and -184) near the active site of the enzyme, we tested whether pretreatment of plasma with the reversible sulfhydryl modifying compound, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), could protect LCAT from CS-induced inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcysteine↗
PubMed1985

Unique electronic property of a Langmuir-Blodgett film of dioleyl lecithin deposited on a porous membrane.

The current-controlled voltage characteristics across a Langmuir-Blodgett film of dioleyl lecithin deposited on a fine-pore membrane between equimolar aqueous solutions of NaC1 and KC1 were studied. This membrane was found to exhibit the properties of switching and differential negative-resistance. These unique electronic properties are interesting in relation to excitable phenomena in biomembranes.

Electrochemistry↗
PubMed1987

The catalytic center of lecithin:cholesterol acyltransferase: isolation and sequence of diisopropyl fluorophosphate-labeled peptides.

Lecithin:cholesterol acyltransferase (LCAT) was purified from hog plasma and subsequently reacted with [3H]-Diisopropyl fluorophosphate (DFP). The labeled enzyme was digested with pepsin and the peptides separated by high performance liquid chromatography (HPLC). Two radioactive peptides were isolated, subjected to automated amino acid sequencing and yielded the following data: A) Ile-Ser-Leu-Gly-Ala-Pro-Trp-Gly-Gly-Ser, and B) Tyr-Ile-Phe-Asp-x-Gly-Phe-Pro-Tyr-x-Asp-Pro-Val. Both of these sequences represent very highly conserved regions of the enzyme when compared to the sequence of human LCAT. Peptide (A) is considered to represent the catalytic center of LCAT based on comparisons with data reported in the literature.

Amino Acid Sequence↗
PubMed1992

An amino acid exchange in exon I of the human lecithin: cholesterol acyltransferase (LCAT) gene is associated with fish eye disease.

The exons of the lecithin:cholesterol acyltransferase (LCAT) gene in DNA samples from two of the original Swedish Fish Eye Disease patients have been amplified by polymerase chain reactions and sequenced by the dideoxy method. The two patients apparently were unrelated. In both patients a mutation in codon 10 of the first exon was found, altering proline10 to leucine. We note that the mutations causing Fish Eye Disease as well as those causing classical LCAT deficiency are spread over most of the translated gene. Why these various mutations in the same gene give rise to two different disease phenotypes remains unexplained.

Base Sequence↗
PubMed1985

A simplified procedure for the in vitro assay of the initial linear rate of the reaction of lecithin-cholesterol acyltransferase in human serum.

A simple sensitive method for the determination of the initial rate of the reaction of lecithin-cholesterol acyltransferase by equilibrating [3H]cholesterol with unesterified cholesterol of human serum is described. The resulting serum is incubated for various time periods at 37 degrees C and the increase of the label in the cholesterol ester fraction is measured. The labeling is effected by a filter paper method in which a paper strip containing the labeled cholesterol is placed in serum at 4 degrees C, thereby preventing the formation of labeled cholesterol esters by the action of the enzyme. The rate of the reaction was linear up to 30 min.

Acetone↗
PubMed1979

Synthesis of lecithin (phosphatidylcholine) from phosphatidylethanolamine in bovine brain.

Choline molecules are needed for the synthesis of acetylcholine and phospholipids in the mammalian brain. An enzymatic activity capable of forming lecithin (phosphatidylcholine) from the step-by-step methylation of phosphatidylethanolamine is identified in the bovine brain. This enzyme(s), phosphatidylethanolamine-N-methyltransferase (EC 2.1.1.17), is localized in the synaptosomal fraction of bovine caudate nucleus, uses S-adenosylmethionine as the methyl donor (apparent Km = 20 micrometers), and has a Vmax of 50--60 pmol/mg protein X h (i.e. about 1% of that found in rat liver). The brain may be able to meet some of its choline requirements by de novo synthesis.

Animals↗
PubMed1991

Comparison of perturbation effect of propranolol, verapamil, chlorpromazine and carbisocaine on lecithin liposomes and brain total lipid liposomes. An EPR spectroscopy study.

Effect of verapamil, propranolol, chlorpromazine and carbisocaine on dynamics and/or order of liposomes (perturbation effect), prepared from different molar ratios of lecithin (PC) and rat brain total lipids (TL) was studied by EPR spectroscopy using spin probes 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The PC liposomes had higher dynamics and/or lower order than the TL liposomes. The perturbation effect of the drugs depended largely on the lipid composition of the liposomes. The drugs at the drug/lipid molar ratios from 0.1 to 1 increased membrane dynamics and/or decreased membrane order. The drugs had the most pronounced perturbation effect in the liposomes prepared from brain total lipids. The effect of the drugs decreased with decreasing the TL/PC ratio in the liposomes and was lowest, almost diminished, in the PC liposomes. Increasing concentration of the drugs decreased the difference between the dynamics and/or order of the PC and TL liposomes and so eliminated the influence of lipid composition on these membrane parameters. The results emphasize the role of lipid composition in studies concerning drug-lipid interactions in model and biological membranes.

Animals↗
PubMed1975

Phosphorus-31 studies on lecithin in ethanol solutions.

31P relaxation times of lecithin in ethanol solutions have been measured in dependence on temperature and water concentration. Trial calculations have been carried out on the assumption of a 2-site exchange model. The results suggest first, the relaxation behaviour is determined by various motional and exchange processes; second, at 29 MHz the dipole-dipole interaction between 31P and protons provides the dominant contribution; third, in general we are not concerned with the case of "extreme narrowing". Morover, there are no negligible intermolecular contributions to relaxation.

Calorimetry↗
PubMed1975

An ESR Study of the mobility of the cholestane spin label in oriented lecithin-cholesterol multibilayers.

The motion of the cholestane spin label in oriented lecithin-cholesterol multibilayers is described in terms of a rotational diffusion about the long molecular axis with diffusion coefficient D parrell and a restricted random librational motion about axes perpendicular to the long axis with diffusion coefficient D1. The diffusion coefficients have been determined from the angular dependence of the ESR line shape at various temperatures and cholesterol contents. The temperature dependence of D parrell and D1 clearly shows the transition from the gel to liquid crystalline phase. Increasing amounts of cholesterol reduce the transition temperature. A strong reduction is found from o to 10 mole % cholesterol. At 50 mole % no longer a sharp transition is observed. In the temperature range from 40 to 80 degrees C the range of D is about 10 times larger than the range of D parrell, indicating a high activation energy for the librational motion arising from a strong hindrance by interaction with surrounding molecules. Cholesterol contents up to 10-20 mole % give an increase of D parrell and D1, arising from strong decrease of the transition temperature in this range. Above 10-20 mole % a reduction of D parrell and D1 is found. However, the effect of cholesterol is much stronger on D1 than on D parrell. In the liquid crystalline phase at about 60 degrees C the effect of cholesterol on D parrell is even negligible, while D1 strongly changes. This indicates that in the liquid crystalline phase only the librational motion is influenced by cholesterol, due to a denser packing of the molecules in the bilayer.

Binding Sites↗
PubMed1975

A 31P NMR study of the thermal transition of dipalmitoyl lecithin vesicles.

Phosphorous-31 nuclear magnetic resonance (31P NMR) was used to study the liquid crystalline transition of sonicated dipalmitoyl lecithin vesicles in D2O. Linewidths were dependent upon temperature and changed dramatically through the transition region. The potential usefulness of 31P NMR spectroscopy for probing the thermal behavior of phospholipid membranes is evaluated and discussed.

Magnetic Resonance Spectroscopy↗
PubMed1975

Cholesterol: lecithin association at molecular ratios of up to 2 : 1.

X-ray diffraction studies of cholestol: egg lecithin mixtures have demonstrated that single phase systems with molecular ratios of up to 2 : 1 can be prepared from solutions in chloroform but that mixtures prepared from ethanol solutions form a single phase only up to a maximum molecular ratio of 1 : 1. The low angle X-ray patterns of the two mixtures (2 : 1 and 1 : 1) are quite distinctive but there is only a small difference in the wide angle spacings. Independent cholesterol reflections begin to appear in the X-ray diffraction pattern of the 2 : 1 mixture after a few days even when the dry sample is contained in a sealed glass capillary tube. Addition of water greatly accelerates this process. In contrast, a 2 : 1 mixture prepared from chloroform solutions can be maintained in sonicated dispersions in water for long periods.

Binding Sites↗
PubMed1976

An ESR study of the anchoring of spin-labeled stearic acid in lecithin multilayers.

In egg lecithin-water lamellar phases, spin-labeled stearic acid gives two superimposed ESR spectra which are only well resolved when the temperature is greater than 30 degrees C. These two spectral components are attributed to the dissociated and non-dissociated forms of the fatty acid carboxylic group, anchored at two different positions in the polar interface constituted by the hydrated lipid polar heads. Results on such interactions of other functional groups (spin-labeled fatty ester and fatty alcohol) are also presented.

Binding Sites↗