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PubMed1977

Surface potential effects on metal ion binding to phosphatidylcholine membranes 31P NMR study of lanthanide and calcium ion binding to egg-yolk lecithin vesicles.

31P NMR of phosphatidylcholine (lecithin) from egg-yolk in sonicated vesicles has been measured in the presence of various ions. Addition of Ln3+ or Ca2+ shifted the 31P resonance of the phosphate groups of the outer surface of the vesicles. These shifts were measured at varied lanthanide or Ca2+ concentration at different ionic strengths obtained by addition of NaCl. The shifts induced by Tb3+ and Ca2+ have been analyzed using the theory of the diffuse double layer. Corrections were introduced for the effect of the ionic strength on the activities of the ions. The binding efficiency is shown to be controlled by the electrostatic potential produced by the bound cations at the membrane surface. This potential is slightly modified due to weak chloride binding. Binding constants have been derived.

Binding Sites↗
PubMed1990

Lamellar packing of a chiral N,N-dimethylphosphatidylethanolamine: electron diffraction. Evidence for a lecithin-type headgroup conformation.

Lamellar electron diffraction intensity data from epitaxially crystallized 1,2-dipalmitoyl-sn-glycerophospho-N,N-dimethylethanolamine were used to determine the layer packing in order to compare the chiral structure to the crystal structure of a racemic homologue. After finding the chain orientation, the structure was determined by interpretation of the Patterson function, followed by independent crystallographic phase assignments with conventional direct methods (use of three phase structure invariants). The phase determination was verified by a translational search with a molecular model based on a similar lecithin structure. The final R-value is 0.29, and this is lowered to 0.18 after a correction is made for incoherent multiple electron scattering. The layer packing is found to be very much like that of a diacyl phosphatidylcholine with the N,N-dimethylethanolamine moiety parallel to the bilayer surface rather than the perpendicular arrangement of headgroups involved in an interdigitated layer, as seen for racemic homolog.

Crystallization↗
PubMed1992

Spontaneous domain formation of phospholipase A2 at interfaces: fluorescence microscopy of the interaction of phospholipase A2 with mixed monolayers of lecithin, lysolecithin and fatty acid.

Fluorescence microscopy has recently been proven to be an ideal tool to investigate the specific interaction of phospholipase A2 with oriented substrate monolayers. Using a dual labeling technique, it could be shown that phospholipase A2 can specifically attack and hydrolyze solid analogous L-alpha-DPPC domains. After a critical extent of monolayer hydrolysis the enzyme itself starts to aggregate forming regular shaped protein domains (Grainger et al. (1990) Biochim. Biophys. Acta 1023, 365-379). In order to confirm that the existence of hydrolysis products in the monolayer is necessary for the observed aggregation of phospholipase A2, mixed monolayers of D- and L-alpha-DPPC, L-alpha-lysoPPC and palmitic acid in different ratios were examined. The phase behavior and the interaction of these films with phospholipase A2 were directly visualized with an epifluorescence microscope. Above a certain critical concentration of lysolecithin and palmitic acid in the monolayer, compression of these mixed films leads to phase separation and formation of mixed domains of unknown composition. Their high negative charge density is evidenced by preferential binding of a cationic dye to these phase-separated areas. Introduction of fluorescence-labeled phospholipase A2 underneath these mixed domains results in rapid binding of the protein to the domains without visible hydrolytic activity, regardless of whether the L-form or the D-form of the DPPC were used. In binary mixtures, only those with DPPC/palmitic acid show formation of phase-separated areas which can be specifically targeted by phospholipase A2 leading to a rapid formation (within 2 min) of protein domains. Experiments with pyrenedecanoic acid containing monolayers give the first direct evidence that acid is located above the enzyme domains. These results show that a locally high negative charge density of the phase-separated domains is one of the prerequisites for the binding of phospholipase A2. In addition, however, small amounts of D- or L-alpha-DPPC headgroups within the domains of the monolayer seem to be necessary for recognition followed by fast binding of the protein to the domains. This is confirmed by experiments with mixed monolayers of diacetylene carboxylic acid and D-alpha-DPPC. The acid--immiscible with lecithin--forms well defined pure acid domains in the monolayer. While the cationic dye can be docked rapidly to these phase separated areas, no preferential enzyme binding and thus no protein domain formation below these acid domains can be induced.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Anomalous phase behavior of long chain saturated lecithin bilayers.

X-ray scattering has been performed on fully hydrated unoriented multilamellar vesicles of lecithins with even chain lengths n from 16 to 24 as a function of temperature in chain ordered phases. The longer chain lengths, n > or = 20, show anomalous behavior compared to the shorter chain lengths, n < 20. This report concentrates on n = 24. Although the history and time dependence shows that equilibrium was not always achieved, it appears that there is a second gel-like phase G2 below 40 degrees C. The G2 phase has a small tilt angle and opposite hexagonal symmetry breaking from the usual G1 gel phase. Also, as T is raised above 45 degrees C, the wide-angle data suggest the appearance of a phase with hexagonal chain packing and small chain tilt angle.

Lipid Bilayers↗
PubMed1976

[Beta-Hydroxybutyrate dehydrogenase of rat liver inner mitochondrial membrane. Its isolation, characterization, and reactivation by lecithins differing in their apolar regions. The influence of the addition of cholesterol on its level of reactivation (author's transl)].

The beta-hydroxybutyrate dehydrogenase has been isolated and purified from the inner mitochondrial membrane of the rat liver. It consists in a dimer of molecular weight 77 000 composed by two subunits of molecular weight 38 000 each. The level of its reactivation by lecithin is influenced by the length and degree of unsaturated of their aliphatic chains. The addition of cholesterol inhibits the reactivation.

Animals↗
PubMed1979

Purification of human plasma lecithin:cholesterol acyltransferase and its specificity towards the acyl acceptor.

A simple and convenient method for the purification of human plasma lecithin-cholesterol acyltransferase was developed. The method involves the adsorption of the enzyme from diluted human plasma on DEAE-Sephadex, treatment with 1-butanol in the presence of (NH4)2SO4, DEAE-Sephadex chromatography, treatment with dextran sulfate in the presence of Ca2+, and hydroxyapatite chromatography. The enzyme purified showed a single main band by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In addition, the enzyme obtained was stable for more than four weeks, when it was kept at 4 degrees C under N2 in a buffer of low ionic strength. The purified enzyme was used to study its specificity toward the acyl acceptor. This specificity was found to be broad in that not only sterols but also long chain primary alcohols exhibited considerable acceptor activity. Furthermore, in agreement with our previous observations with crude enzyme (Piran, U. and Nishida, T. (1976) J. Biochem. (Tokyo) 80, 887-889), the purified enzyme was found to be capable of hydrolyzing the ester linkage at the carbon-2 position of phosphatidylcholine. The transesterification, as well as the hydrolytic reaction, required the presence of the cofactor polypeptide, apolipoprotein A-I.

Apolipoproteins↗
PubMed1980

Influence of lecithin:cholesterol acyltransferase on cholesterol metabolism in hepatoma cells and hepatocytes.

Cholesterol content and synthesis were measured in rabbit hepatocytes and rat hepatoma cells (Fu5AH) incubated in rabbit serum at concentrations ranging from 2.5% to 50%. Values were compared to controls grown in delipidized serum protein. Cellular cholesterol content varied inversely with the serum concentration, whereas cholesterol synthesis was elevated as serum concentration in the incubation medium was raised. The reduction in cellular cholesterol content and the elevation in synthesis observed with the cells incubated in high concentrations of fresh serum could be correlated with the extent of serum lipoprotein modification by lecithin:cholesterol acyltransferase. Unmodified serum in which LCAT had been inactivated depressed cholesterol synthesis and increased cellular cholesterol content at all concentrations. The presence of active LCAT was not required for the cellular responses, since serum which had been modified before LCAT inactivation also stimulated cholesterol synthesis and decreased content. Qualitatively similar results were obtained with human, rat and rabbit sera. Fractionation of serum demonstrated that the stimulatory activity of LCAT-modified serum was associated primarily with the high-density lipoprotein fraction. Comparative cholesterol flux studies using prelabeled hepatoma cells exposed to either normal or modified high-density lipoproteins demonstrated that cellular cholesterol efflux was somewhat depressed in the presence of the modified lipoprotein whereas cholesterol influx was markedly reduced. These data indicate that LCAT modification of serum lipoproteins alters the relative rates of cholesterol flux with the major effect being on cholesterol uptake. This results in a net loss of cholesterol from the cells accompanied by a stimulation of cholesterol synthesis.

Acetates↗
PubMed1988

Molecular cloning of a phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Sequence homologies with lecithin-cholesterol acyltransferase and other lipases.

We have determined the nucleotide sequence of a gene encoding Aeromonas hydrophila phospholipid-cholesterol acyltransferase, an enzyme which shares many properties with mammalian lecithin:cholesterol acyltransferase. The derived amino acid sequence of the protein contains two regions which are homologous to the proposed active sites and binding sites of the plasma acyltransferase and to similar sequences in other interfacially acting lipolytic enzymes. The amino terminus is preceded by a typical 18 amino acid signal sequence. The protein, which is released into the culture supernatant by Aeromonas hydrophila, is confined to the periplasm of Escherichia coli.

Acyltransferases↗
PubMed1988

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma.

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.

Adult↗
PubMed1988

Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I.

To study the activation of lecithin-cholesterol acyl transferase (LCAT) (phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) by apolipoprotein D in comparison to apolipoproteins A-I and C-I, proteoliposomes with a phosphatidylcholine/free cholesterol molar ratio of 24:1, containing 10-300 micrograms/ml of apolipoproteins were used. The proteoliposomes were prepared by the cholate dialysis technique. In all proteoliposome preparations we found rouleaux structures and stacked discs. The particles formed with apolipoprotein A-I were the most homogeneous, followed by apolipoprotein D- and apolipoprotein C-I-containing particles. Apolipoprotein A-I was the most potent LCAT activator in our system followed by apolipoproteins C-I and D. The fractional esterification rate observed with apolipoprotein D-containing substrates amounted to 15-48% that of apolipoprotein A-I-containing ones. Neither apolipoprotein A-I- nor C-I-containing proteoliposomes gave linear reaction kinetics with LCAT. Even during the first 15-30 min of incubation, the kinetics deviated strikingly from linearity at all apolipoprotein concentrations. In contrast, proteoliposomes containing apolipoprotein D exhibited linear reaction kinetics up to 60-90 min. At low apolipoprotein A-I concentrations (5 micrograms/ml), the addition of apolipoprotein D to the incubates resulted in significantly higher esterification rates as compared to substrates containing apolipoprotein A-I only. This was not the case using substrates with high apolipoprotein A-I concentrations (50 micrograms/ml). From our results we speculate that apolipoprotein D may have some stabilizing effect on the enzyme LCAT.

Amino Acids↗
PubMed1989

Reactivity of HDL subfractions towards lecithin-cholesterol acyltransferase. Modulation by their content in free cholesterol.

(1) Human HDL2 (d 1.070-1.125) and HDL3 (d 1.125-1.21) labelled with unesterified [14C]cholesterol, were incubated with a source of lecithin-cholesterol acyltransferase. For optimal activity, the reaction required the addition of albumin in excess, at least 3-times greater than the concentration of HDL-free cholesterol. Under such conditions, the reaction appeared saturable. HDL3 was found the most efficient substrate and the Vmax values expressed for 1.5 IU LCAT/ml and with an albumin/free cholesterol ratio of 3, were 8.3 nmol free cholesterol esterified/ml per h and 4.1 nmol/ml per h for HDL3 and HDL2, respectively. (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis with a semipurified lipoprotein lipase from bovine milk. The newly formed HDL had gained free cholesterol and phospholipids, so that about 50% of these modified HDL, referred to as light-LIP-HDL3, were reisolated in the HDL2 density range. Light-LIP-HDL3 were enriched mostly in free cholesterol (+ 160%) and in phospholipid (+ 40%). Their reactivity towards LCAT was half-reduced compared to parent HDL3, which correlated well with a decrease in their phospholipid/free cholesterol molar ratio. Moreover, HDL3 artificially enriched in free cholesterol and exhibiting a comparable PL/FC behaved like lipolysis-modified HDL in their reactivity towards LCAT. (3) HDL3 were also modified by co-incubation with VLDL (post-VLDL-HDL3), or with VLDL and a source of lipid transfer protein (CET-HDL3). The latter treatment greatly affected the lipid composition of the core particle (-25% esterified cholesterol, +190% TG). In both cases, the moderate decreasing LCAT reactivity observed could be related to the phospholipid/free cholesterol ratio. Thus, like in artificial substrates, the lipid composition of the HDL surface may control the rate of LCAT-mediated cholesterol esterification.

Animals↗
PubMed1989

Effect of intralipid infusion on lecithin:cholesterol acyltransferase and lipoprotein lipase in young rats.

We compared the effects of Intralipid and dextrose infusion on plasma lecithin:cholesterol acyltransferase (LCAT), plasma lipid profiles and lipolytic activity. We used 5-week-old male Sprague-Dawley rats which were given total parenteral nutrition (TPN) with either Intralipid (3 g/kg body weight) or an equicaloric amount of 25% dextrose in the presence or absence of heparin (1 or 10 IU/ml of TPN). 40 min after the end of 4 h of infusion, plasma LCAT activity was significantly decreased (P less than 0.001), while total cholesterol and free fatty acid levels were significantly (P less than 0.05) increased in rats given Intralipid as compared to those given dextrose. We found associations (P less than 0.005) between LCAT activity and total cholesterol and between LCAT and free fatty acid levels; the coefficients of negative correlation were 0.543 and 0.607, respectively. Concomitantly to the increment in plasma total cholesterol levels, there was a decrease in the high-density lipoprotein (HDL) cholesterol fraction; the latter, which was 40% of the total plasma cholesterol in control and dextrose-infused rats, declined to 9% in rats given Intralipid. Administration of heparin during Intralipid infusion, even up to 10 IU/ml of TPN, did not affect any of these changes. After dextrose infusion, the values of all three parameters were similar to those of the control group. Plasma lipolytic activity was not significantly different between rats given infusion (Intralipid or dextrose) and controls. However, in the presence of heparin, plasma lipolytic activity increased similarly in both infused groups. These data indicate that in young rats, Intralipid infusion leads to an increase in plasma total cholesterol and free fatty acid levels, which correlates with a decrease in LCAT activity; the concurrent decrease in HDL cholesterol levels might account, in part, for the loss of LCAT activity. The administration of heparin results in an elevation of plasma lipolytic activity; however, it does not prevent the hypercholesterolemia, nor the decline in LCAT activity associated with Intralipid infusion.

Animals↗
PubMed1990

Studies on the lecithin: cholesterol acyltransferase substrate properties of HDL as determined by its subclass distribution analysed by gradient gel electrophoresis.

In order to study the impact of high-density lipoproteins (HDL) subclasses on the ability of HDL to act as substrate for lecithin: cholesterol acyltransferase (LCAT), we isolated HDL from nine normolipidemic male subjects. The HDL particle size distribution was analysed by gradient gel electrophoresis and the esterification rate of the isolated homologous HDL was compared with a pool of HDL where all the nine subjects took part. It was found that the strongest determinant for HDL cholesterol esterification rate was the inhibitory action of HDL subclass 2B.

Adult↗
PubMed1991

Plasma lecithin-cholesterol acyltransferase activity and cholesterol and phospholipid levels in premature newborn infants.

Lecithin-cholesterol acyltransferase (LCAT) activity has been suggested to play an important role in the regulation of lipid metabolism. The present study was undertaken to examine any relationship between LCAT activity and altered cholesterol levels in plasma of full-term and preterm newborn infants. Plasma total, free and esterified cholesterol, total phospholipid and LCAT activity (cholesterol esterified, nmol/ml per h) were determined in placental cord blood. There was a significant negative relationship between total cholesterol levels and gestational age. The increased cholesterol with prematurity was due to both free and esterified cholesterol. There was also a significant negative relationship between LCAT activity and free cholesterol levels but not between LCAT activity and total cholesterol and esterified cholesterol levels. There was no relationship between esterified-to-free cholesterol ratio and LCAT activity. Total phospholipid was not significantly related to either gestational age or LCAT activity. This study suggests that reduced LCAT activity may be one of the factors that result in the accumulation of cholesterol in premature infants.

Cholesterol↗
PubMed1992

Differential effect of subspecies of lipoprotein containing apolipoprotein A-I on cholesterol efflux from cholesterol-loaded macrophages: functional correlation with lecithin: cholesterol acyltransferase.

Two species of lipoprotein containing apoA-I, one containing only apoA-I (LpA-I), and the other containing apoA-I and apoA-II (LpA-I/A-II), were tested for their effects on macrophage foam cells. Rat macrophages were converted to foam cells by incubation with radiolabeled acetylated LDL. Incubation with LpA-I or LpA-I/A-II decreased the cellular cholesteryl esters (CE) mass. However, the free cholesterol (FC) mass was only reduced by LpA-I. All the radioactivity excreted into the medium was associated with LpA-I or LpA-I/A-II; 39% of the excreted radioactivity was esterified in LpA-I and 10% in LpA-I/A-II. Upon complete inactivation of lecithin: cholesterol acyltransferase (LCAT) activity with dithiobisnitrobenzoic acid, the cholesterol reducing capacity of LpA-I was weakened significantly. However, the CE mass reducing capacity of LpA-I/A-II was not affected. When LpA-I and LpA-I/A-II were combined, the cholesterol reducing capacity of the mixture was similar to that of LpA-I alone. However, LpA-I re-isolated from the medium showed a lower esterification rate than did the re-isolated LpA-I/A-II, thereby indicating that the cholesterol esterified in LpA-I was transferred to LpA-I/A-II. These results suggest that (i) the function of LpA-I is closely linked to the LCAT activity while that of LpA-I/A-II is not, and (ii) LpA-I in concert with LpA-I/A-II induces a series of extracellular events; LCAT-mediated esterification of excreted FC by LpA-I and a subsequent CE transfer to LpA-I/A-II. These mechanisms might be important for net cholesterol efflux from macrophage foam cells in physiological states.

Adolescent↗
PubMed1992

Altered positional specificity of human plasma lecithin-cholesterol acyltransferase in the presence of sn-2 arachidonoyl phosphatidyl cholines. Mechanism of formation of saturated cholesteryl esters.

The positional specificity of purified human lecithin-cholesterol acyltransferase (LCAT) was studied by analyzing the labeled cholesteryl ester (CE) species formed in the presence of proteoliposome substrates containing mixed chain phosphatidylcholine (PC) species, labeled cholesterol and apoprotein A-I. Whereas over 90% of the acyl groups used for CE synthesis were derived from the sn-2 position of most of the naturally occurring PC substrates, about 75% of the CE species formed in the presence of sn-1-myristoyl 2-arachidonoyl PC, sn-1-palmitoyl-2-arachidonoyl (PAPC) and sn-1-palmitoyl 2-docosahexaenoyl PC were derived from the sn-1-position. On the other hand, rat LCAT utilized mostly sn-2-acyl group from either PAPC or from sn-1-palmitoyl 2-linoleoyl PC. The positional specificity of the human enzyme was not affected by the alteration in the matrix fluidity, type of the apoprotein activator used, or by the free cholesterol/PC ratio in the substrate. These results show that the positional specificity of human plasma LCAT is altered in the presence of sn-2-arachidonoyl PC, or sn-2-docosahexaenoyl PC, probably due to steric restrictions at the active site, and this may account for the formation of disproportionately high concentrations of saturated CE, and low concentrations of long-chain polyunsaturated CE in human plasma, relative to the composition of sn-2-acyl groups in plasma PC.

Animals↗
PubMed1992

Opposite regulation of hepatic lipase and lecithin: cholesterol acyltransferase by glucocorticoids in rats.

Rats were treated with hydrocortisone, dexamethasone or triamcinolone for 4 days. The effect of treatment on hepatic lipase and lecithin:cholesterol acyltransferase (LCAT) mRNA levels and catalytic activities was determined. Hepatic lipase mRNA was not affected by hydrocortisone, but was decreased after dexamethasone (-28%) and triamcinolone (-54%). Hepatic lipase activity followed the same pattern, it was not affected by hydrocortisone and lowered by dexamethasone (-38%) and triamcinolone (-70%). The LCAT mRNA level in the liver was also not affected by hydrocortisone, but increased upon treatment with dexamethasone (+22%) and triamcinolone (+72%). Plasma LCAT, determined with an excess exogenous substrate (designated LCAT-II), tended to decrease after hydrocortisone treatment (-11%) and was higher after dexamethasone (+21%) and triamcinolone (+22%). The plasma cholesterol esterification rate (designated LCAT-I), determined by incubation of the plasma at 37 degrees C, followed the same pattern. The activity ratio of hepatic lipase/LCAT-II decreased from 1 in the controls to 0.51 after dexamethasone and 0.25 in the triamcinolone-treated animals. The plasma HDL cholesterol concentration in the different groups changed oppositely to the hepatic lipase/LCAT activity ratio. It is concluded that HDL cholesterol is raised by synthetic glucocorticoids due, among other factors, to a lowered hepatic lipase and an increased plasma LCAT activity. The influence of glucocorticoids on these enzymes is, at least partly, explained by the effects on the hepatic mRNA contents.

Animals↗
PubMed1993

Lecithin-cholesterol acyltransferase: effects of mutagenesis at N-linked oligosaccharide attachment sites on acyl acceptor specificity.

Site-directed mutagenesis was used to generate lecithin-cholesterol acyltransferase (LCAT) species in which individual attachment sites for N-linked oligosaccharide residues were replaced with residues that prevent the attachment of carbohydrate. Mutants at three of four sites retained significant acyltransferase activity, and phospholipase activity in the absence of cholesterol. Mutation at one site (asn272) converted LCAT to a phospholipase generating fatty acids not cholesteryl esters.

Amino Acid Sequence↗