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PubMed1980

Membrane phospholipid synthesis in Escherichia coli. Identification of the sn-glycerol-3-phosphate acyltransferase polypeptide as the plsB gene product.

A collection of hybrid plasmids bearing a structural gene, plsB, for the sn-glycerol-3-phosphate acyltransferase of Escherichia cole (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) J. Biol. Chem. 255, 9413-9420) was employed to identify the membrane protein which is the sn-glycerol-3-phosphate acyltransferase. Strains containing these hybrid plasmids exhibited a marked increase in sn-glycerol-3-phosphate acyltransferase activity which was quantitatively extracted from membrane preparations with Triton X-100. Analysis of polypeptides present in detergent extracts of membranes from strains harboring the hybrid plasmids revealed a marked overproduction of a protein with an apparent molecular weight of 83,000, which was also the major protein labeled in minicells containing these hybrid plasmids. The labeled 83,000-dalton protein cochromatographed with sn-glycerol-3-phosphate acyltransferase activity on DEAE-cellulose. Utilization of three hybrid plasmids bearing amber mutations within the plsB gene demonstrated that the 83,000-dalton protein is the sn-glycerol-3-phosphate acyltransferase. Analysis of Bam HI deletion plasmids demonstrated that a 2.3-megadalton DNA fragment is necessary and sufficient for expression of the plsB gene. The sn-glycerol-3-phosphate acyltransferase was purified to near homogeneity from Triton X-100 extracts of membranes from overproducing strains. The preparations had reconstitutable specific activity of 2.5 micromol/min/mg and contained a single polypeptide with an apparent molecular weight of 83,000.

Acyltransferases↗

PubMed1982

Regulation of rabbit intestinal acyl coenzyme A-cholesterol acyltransferase in vivo and in vitro.

The rate of intestinal cholesterol esterification may be an important determinant of the rates of entry and exit of cholesterol from the body. Acyl coenzyme A-cholesterol acyltransferase, the intracellular cholesterol esterifying enzyme, may play a role in this process. To assess this, the response of rabbit intestinal acyl coenzyme A-cholesterol acyltransferase in vivo was studied. Animals were fed a diet containing cholesterol and corn oil, and they responded with an increase in acyl coenzyme A-cholesterol acyltransferase activity. The increase was apparent in all segments of the intestine proximal to the distal ileum, and it occurred specifically in the villus cells where the bulk of lipid absorption is believed to take place. In cultured intestinal explants, the activity responded rapidly to sterols (increased) and to fatty acids (decreased) when control intestine was used. If intestine from cholesterol-corn oil-fed animals was cultured, sterols still induced an increase, but fatty acids did not affect the enzyme activity. The acutely induced increases in acyl coenzyme A-cholesterol acyltransferase activity were not prevented by cycloheximide. The results show that acyl coenzyme A-cholesterol acyltransferase in the absorptive cells of intestine responds both acutely and chronically to dietary factors, supporting the hypothesis that acyl coenzyme A-cholesterol acyltransferase plays a role in cholesterol absorption.

Acyltransferases↗

PubMed2003

Stimulation of rat liver mitochondrial sn-glycerol-3-phosphate acyltransferase by polymyxin B via enhanced extraction of lysophosphatidic acid.

The purpose of this investigation was to determine how polymyxin B stimulates the activity of mitochondrial glycerophosphate acyltransferase. Polymyxin B did not change the integrity of the mitochondrial outer membrane as judged by testing the latency (>80%) of cytochrome oxidase activity. The stimulation totally disappeared when polymyxin B-treated mitochondria were washed. The FA side chain in polymyxin B was unnecessary for stimulation, as the nonapeptide was as effective as the whole antibiotic. The stimulation by polymyxin B or the nonapeptide was observed only in the presence of BSA. Cytochrome c, when added to the incubation medium instead of albumin, did not stimulate the mitochondrial enzyme, but did produce a stimulatory effect of polymyxin B on the mitochondrial acyltransferase. As reported earlier for the bacterial and microsomal acyltransferase, other polycationic compounds such as spermine and spermidine stimulated mitochondrial glycerophosphate acyltransferase. The stimulation of the mitochondrial acyltransferase by spermine and spermidine also occurred only in the presence of BSA. The analysis of the products of esterification demonstrated the presence of more lysophosphatidic acid (LPA) in the polymyxin B- and polyamine-stimulated assays in comparison to their respective control. Furthermore, in comparison to the albumin-treated control, there was 60% more LPA present in the assay supernatant fractions of polymyxin B-treated samples. Our results suggest that polymyxin B stimulates the mitochondrial glycerophosphate acyltransferase activity by enhancing the extraction of more LPA from the mitochondria to the supernatant fraction.

Acylation↗

PubMed1982

Evidence that reverse cholesterol transport occurs in vivo and requires lecithin-cholesterol acyltransferase.

The transport of cholesterol from extrahepatic tissues into plasma (reverse cholesterol transport) and the possible requirement for lecithin:cholesterol acyltransferase was examined in the rat. One hour after removal of the liver plasma cholesterol ester concentrations were significantly increased by 20%, whereas free cholesterol concentrations were unchanged. The lecithin:cholesterol acyltransferase inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was administered to eviscerated rats. It inhibited plasma lecithin:cholesterol acyltransferase activity by 90% which in turn totally prevented the increase in plasma cholesterol ester concentrations. In addition, heat-inactivated plasma from DTNB-treated eviscerated rats was 50% more reactive toward a standard source of lecithin:cholesterol acyltransferase compared to plasma from control or untreated eviscerated rats. These data suggest that in the rat a reactive lecithin:cholesterol acyltransferase substrate is formed extrahepatically. Together with lecithin:cholesterol acyltransferase, this reactive substrate removes cholesterol from peripheral tissues.

Animals↗

PubMed1982

Study of the lecithin: cholesterol acyltransferase reaction with liposome and high density lipoprotein substrates.

The activity of highly purified preparations of human plasma lecithin: cholesterol acyltransferase were stabilized by precipitating the enzyme with ammonium sulfate and using the dilutions of the particulate lecithin: cholesterol acyltransferase suspension for enzyme assays. Ammonium sulfate concentrations in the assay mix up to 0.1 M had no significant effect on lecithin: cholesterol acyltransferase activity. The basic enzymatic properties of lecithin: cholesterol acyltransferase were investigated using liposomes and high density lipoprotein (HDL) substrates. pH optima for both substrates was approximately 8.0. The temperature dependence of lecithin: cholesterol acyltransferase activity resulted in non-linear Arrhenius plots with both substrates. The activity vs. temperature (degrees C) curves showed slight inflections at 30 degrees C, which may have been due to the relatively rapid inactivation of the enzyme above this temperature. HDL3 was found to be a better substrate than HDL or HDL2. HDL3 was also considerably better than egg phosphatidylcholine/cholesterol liposomes as an lecithin: cholesterol acyltransferase substrate. Addition of HDL2 to a reaction mix of enzyme and HDL3 indicated that HDL2 acts as an inhibitor of cholesterol esterification in this system.

Apolipoprotein A-I↗

PubMed1983

Evidence that lecithin:cholesterol acyltransferase acts on both high-density and low-density lipoproteins.

In incubations of plasma containing lipoproteins at physiological concentrations it has been confirmed that high-density lipoproteins (HDL) are the major initial recipients of the esterified cholesterol formed in the reaction catalysed by lecithin:cholesterol acyltransferase. It has also been confirmed, however, that a small proportion of the esterified cholesterol of lecithin:cholesterol acyltransferase origin is incorporated directly into low-density lipoproteins (LDL), via a pathway that bypasses the HDL. This direct incorporation of esterified cholesterol into LDL is compatible with either of two general models. Model A proposes that lecithin:cholesterol acyltransferase does not interact directly with LDL but rather that it acts only on lipoproteins outside the LDL fraction. According to model A, while most of the esterified cholesterol so formed is incorporated into HDL, a small proportion is transferred directly to LDL. Model B, by contrast, proposes that a direct incorporation of esterified cholesterol into LDL is the result of a direct action of lecithin:cholesterol acyltransferase on the free cholesterol associated with LDL. To differentiate between these two models, experiments have been performed in which incubation mixtures containing LDL, HDL and a source of lecithin:cholesterol acyltransferase were supplemented with free [3H]cholesterol which had previously been incorporated into either LDL or HDL. It was found that, of the esterified [3H]cholesterol which was subsequently formed, the proportion recovered in the LDL fraction was much greater in the incubations to which the free [3H]cholesterol had been added as a component of LDL than in those to which it had been added as a component of HDL. This essentially excluded model A but was consistent with model B. It has been concluded that, while most of the lecithin:cholesterol acyltransferase may interact with particles in the HDL fraction, a small proportion of the enzyme interacts directly with LDL.

Adolescent↗

PubMed1985

A rapid large-scale procedure for purification of lecithin-cholesterol acyltransferase from human and animal plasma.

A rapid and convenient procedure was developed for large-scale purification of lecithin-cholesterol acyltransferase from the plasma of humans, pigs, dogs, goats and rabbits. The procedure included dextran sulfate-Mg2+ precipitation, Phenyl-Sepharose column, Affi-gel blue column, DEAE-Sepharose column, DEAE-Affi-gel blue column and hydroxyapatite column. Lecithin-cholesterol acyltransferase was purified approx. 20 000-fold with about 14% yield from human plasma. A similar result of purification was obtained from pigs, dogs, goats and rabbits. The final enzyme preparation from all five mammalian species was homogeneous as judged by SDS-polyacrylamide gel electrophoresis and high-performance gel filtration in the presence of SDS. The apparent molecular weight of the purified enzymes from humans, pigs, dogs, goats and rabbits were 65 000, 66 000, 65 000, 65 000 and 66 000, respectively, as determined by SDS-polyacrylamide gel electrophoresis and were 67 000, 67 000, 66 000, 66 000 and 67 000, respectively, by high-performance gel filtration. Studies with DEAE-Sepharose columns demonstrated that lecithin-cholesterol acyltransferase from these mammalian species was similar in molecular charge. The pH optimum for activity of purified enzyme ranged from 7.4 to 8.0 among these species. All purified lecithin-cholesterol acyltransferase species were activated by human apolipoprotein A-I and were similarly inhibited by p-hydroxymercuribenzoate and phenylmethylsulfonyl fluoride. We concluded that this purification scheme is suitable for rapid isolation of lecithin-cholesterol acyltransferase from the plasma of humans, pigs, dogs, goats and rabbits and, by inference, from other mammalian species. The availability of purified active lecithin-cholesterol acyltransferase from various animal species should facilitate phylogenetic and comparative studies of the enzyme.

Animals↗

PubMed1986

Phospholipase A2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferase reactivity.

Human HDL3 (d 1.125-1.21 g/ml) were treated by an exogenous phospholipase A2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5-10% haematocrit and HDL3 (0.6 mM total cholesterol) from 0 to 12-15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 microM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. (2) The lecithin: cholesterol acyltransferase reactivity of HDL3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21-1.25 g/ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15-30% lipolysis, the lecithin: cholesterol acyltransferase reactivity of HDL3 was reduced about 30-40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. (3) The addition of the lecithin: cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin: cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin: cholesterol acyltransferase-induced removal of cellular cholesterol.

Cholesterol↗

PubMed1986

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA:cholesterol acyltransferase.

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA: cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA: cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA: cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.

Animals↗

PubMed1987

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase.

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.

Binding Sites↗

PubMed1987

Effects of dietary cholesterol and fat, sex and sire on lecithin-cholesterol acyltransferase activity in baboons.

We analyzed the effects of dietary cholesterol, type of dietary fat, sex and sire progeny family on lecithin-cholesterol acyltransferase activity in 80 adult baboons. The animals were the progeny of 80 dams and 6 sires and were randomly assigned at birth to breast feeding or to one of three formulas containing 0.02, 0.30 or 0.60 mg cholesterol/ml. After weaning at 4 months of age the animals were fed one of four diets that were either high or low in cholesterol with 40% of the calories from either saturated or unsaturated fat. The fractional and molar rates of lecithin-cholesterol acyltransferase activity were measured at 7-8 years of age by an HPLC method. Infant diet (breast vs. formula feeding or level of cholesterol in formula had no effect on enzyme activity later in life. The adult diets that were high in cholesterol decreased the fractional lecithin-cholesterol acyltransferase rate by 20% / compared to diets low in cholesterol (7.89 vs. 9.84%/h, P less than 0.002), but dietary cholesterol did not affect the molar activity. Animals fed the high cholesterol diets had higher unesterified cholesterol concentrations compared to those fed the low cholesterol diets (38.1 mg/dl vs. 31.6 mg/dl, P less than 0.0001). The molar lecithin-cholesterol acyltransferase rate was increased 13% by saturated compared to unsaturated fat (83.3 vs. 73.6 nmol/h per ml plasma, P less than 0.07), but no effect of dietary fat was observed on the fractional enzyme activity. Females compared to males had significantly higher fractional (10.9 vs. 7.14%/h, P less than 0.0001) and molar lecithin-cholesterol acyltransferase activities (99.3 vs. 61.7 nmol/h per ml plasma, P less than 0.0001). After adjustment for the effects of diet and sex we observed differences in the fractional activity (range, 7.2-10.8%/h, P less than 0.04) and in the molar rate (range, 63.6-99.8 nmol/h per ml plasma, P less than 0.07) among the six sire progeny groups. The differences among sire progeny groups are evidence for genetic differences in lecithin-cholesterol acyltransferase activities among the baboon families.

Animals↗

PubMed2005

Characterisation of acyltransferases from Synechocystis sp. PCC6803.

As phylogenetic ancestors of plant chloroplasts cyanobacteria resemble plastids with respect to lipid and fatty acid composition. These membrane lipids show the typical prokaryotic fatty acid pattern in which the sn-2 position is exclusively esterified by C(16) acyl groups. In the course of de novo glycerolipid biosynthesis this prokaryotic fatty acid pattern is established by the sequential acylation of glycerol-3-phosphate with acyl-ACPs by the activity of different acyltransferases. In silico approaches allowed the identification of putative Synechocystis acyltransferases involved in glycerolipid metabolism. Functional expression studies in Escherichia coli showed that sll1848 codes for a lysophosphatidic acid acyltransferase with a high specificity for 16:0-ACP, whereas slr2060 encodes a lysophospholipid acyltransferase, with a broad acyl-ACP specificity but a strong preference for lysophosphatidyglycerol especially its sn-2 acyl isomer as acyl-acceptor. The generation and analysis of the corresponding Synechocystis knockout mutants revealed that lysophosphatidic acid acyltransferase unlike the lysophospholipid acyltransferase is essential for the vital functions of the cells.

Acyltransferases↗

PubMed2004

Serine carboxypeptidase-like acyltransferases.

In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1-O-beta-acetal esters (1-O-beta-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of alpha/beta hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.

Acyltransferases↗

PubMed1999

Inhibition of lecithin cholesterol acyltransferase by phosphatidylcholine hydroperoxides.

To gain insight into the nature of the lecithin-cholesterol acyltransferase inhibitory factor(s), we separated and collected the oxidation products from oxidized lipoproteins after lipoxygenase treatment. Isolated fractions identified by chemiluminescence, as hydroperoxides of phosphatidylcholine, were found to produce a significant reduction of lecithin-cholesterol acyltransferase activity. The reaction kinetics of lecithin-cholesterol acyltransferase with reconstitued high density lipoproteins were studied in the presence of 0.6 and 1.2 microM hydroperoxides of phosphatidylcholine. No significant changes in the apparent Vmax were observed but a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values was observed with increasing hydroperoxide concentrations. These results show that the active site of lecithin-cholesterol acyltransferase is not affected by the presence of phosphatidylcholine hydroperoxides. Nevertheless, hydroperoxides of phosphatidylcholine altered the reactivity of lecithin-cholesterol acyltransferase for reconstitued high density lipoproteins suggesting either an alteration of the binding of lecithin-cholesterol acyltransferase to the reconstitued high density lipoproteins or a competitive inhibition mechanism.

Apolipoprotein A-I↗

PubMed1999

Alkyl-dihydroxyacetone phosphate synthase and dihydroxyacetone phosphate acyltransferase form a protein complex in peroxisomes.

Dihydroxyacetone phosphate (GrnP) acyltransferase and alkyl-GrnP synthase are the key enzymes involved in the biosynthesis of ether phospholipids. Both enzymes are located on the inside of the peroxisomal membrane. Here we report evidence for a direct interaction between these enzymes obtained by the use of chemical cross-linking. After cross-linking and immunoblot analysis alkyl-GrnP synthase could be detected in a 210-kDa complex which was located entirely on the lumenal side of the peroxisomal membrane. Two-dimensional SDS/PAGE demonstrated that GrnP-acyltransferase is also cross-linked in a 210-kDa complex. Co-immunoprecipitation confirmed that the two enzymes interact, in a heterotrimeric complex. Furthermore, alkyl-GrnP synthase can form a homotrimeric complex in the absence of GrnP-acyltransferase as was demonstrated by immunoblot analysis after cross-linking experiments with either GrnP-acyltransferase deficient human fibroblast homogenates or recombinant (His)6-tagged alkyl-GrnP synthase. We conclude that alkyl-GrnP synthase interacts selectively with GrnP-acyltransferase in a heterotrimeric complex and in the absence of GrnP-acyltransferase can also form a homotrimeric complex.

Acyltransferases↗

PubMed1985

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV.

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.

Apolipoprotein A-I↗

PubMed1993

The firA gene of Escherichia coli encodes UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. The third step of endotoxin biosynthesis.

The possibility that the firA gene of Escherichia coli (Dicker, I. B., and Seetharam, S. (1991) Mol. Microbiol. 6, 817-823) might function in lipid A biosynthesis was examined based on its homology to the lpxA gene, which encodes UDP-N-acetylglucosamine O-acyl-transferase, the first enzyme in lipid A formation. Extracts of a temperature-sensitive firA mutant, RL-25, were assayed for their ability to acylate UDP-GlcNAc, using a coupled assay. The results suggested that extracts of RL-25 might be defective in the third enzyme of this pathway, the UDP-3-O-(R-3-hydroxymyristoyl)-glucosamine N-acyltransferase. Living cells of RL-25 also displayed a 5-fold decreased rate of lipid A biosynthesis at the nonpermissive temperature as judged by a 32Pi incorporation assay. In order to examine N-acyltransferase activity directly, the substrate [alpha-32P]UDP-3-O-(R-3-hydroxymyristoyl)-GlcN was synthesized enzymatically. N-Acyltransferase specific activity in RL-25 extracts was reduced to less than 10% of wild-type. When the wild-type firA gene was cloned into a T7-based expression vector, N-acyltransferase specific activity increased almost 360-fold relative to wild-type extracts, demonstrating that firA is the structural gene for the enzyme. The N-acyltransferase displays absolute specificity for the R-3-OH moiety of R-3-hydroxymyristoyl-ACP, as does the O-acyltransferase, consistent with the placement of R-3-hydroxymyristate in E. coli lipid A.

Acyltransferases↗

PubMed1977

Properties of dihydroxyacetone phosphate acyltransferase in the harderian gland.

We have used a microsomal preparation from the pink portion of the rabbit harderian gland to study the properties of dihydroxyacetone-P acyltransferase. The enzymatic activity in the microsomes is latent and is stimulated approximately 20-fold by the addition of detergent to the assay system. Both deoxycholate and cholate stimulated the enzyme at concentrations considerably below the critical micellar concentration of these detergents. The acyltransferase was not solubilized by treatment with these detergent concentrations. In addition, we have shown that the acyltransferase is sensitive to proteolytic digestion in the presence of deoxycholate concentrations that render the microsomal vesicles permeable to the protease, but is insensitive to the protease treatment in the absence of detergent. Collectively, these data indicate that the active site of dihydroxyacetone-P acyltransferase is exposed to the lumenal surface of the microsomal vesicles. This enzyme is distinct from the glycerol-P acyltransferase in that the dihydroxyacetone-P acyltransferase is sensitive to protease digestion only in the presence of deoxycholate, is not inhibited by sulfhydryl reagents, and is not competitively inhibited by sn-glycerol-3-P.

Acyltransferases↗