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PubMed2001

The initial step of the glycerolipid pathway: identification of glycerol 3-phosphate/dihydroxyacetone phosphate dual substrate acyltransferases in Saccharomyces cerevisiae.

The initial step of phospholipid biosynthesis in yeast is carried out through the acylation of glycerol 3-phosphate (G-3-P) and dihydroxyacetone phosphate by stereospecific sn-1 acyltransferases. Here we report the identification of two key fatty acyltransferases of the glycerolipid biosynthesis pathway in Saccharomyces cerevisiae. Disruption of the open reading frame YBL011w, corresponding to a gene previously identified as a choline transporter suppressor (SCT1), resulted in a substantial decrease of total cellular G-3-P acyltransferase activity. A yeast strain disrupted at the open reading frame YKR067w, which encodes a protein closely related to Sct1p, also exhibited a dramatic reduction in G-3-P acyltransferase activity. Molecular characterizations of the genes revealed that a missense mutation in YKR067w accounted for a defect in the activities of the G-3-P acyltransferase in the yeast mutant strain TTA1. Heterologous expression of YKR067w in Escherichia coli further confirmed its enzyme activity. These results indicate that YKR067w and YBL011w, designated herein as GAT1 and GAT2(SCT1), respectively, are yeast G-3-P acyltransferase genes. Furthermore, biochemical results are presented to show that both Gat1p and Gat2p(Sct1p) are G-3-P/dihydroxyacetone phosphate dual substrate-specific sn-1 acyltransferases. The fatty acyl specificity of Gat1p is similar to that of the mammalian microsomal G-3-P acyltransferase, as it can effectively utilize a broad range of fatty acids as acyl donors. In contrast, Gat2p(Sct1p) displayed preference toward 16-carbon fatty acids. The most notable of the altered phospholipid compositions of the gat1Delta and gat2(sct1)Delta strains are a decreased phosphatidic acid pool and an increased phosphatidylserine/phosphatidylinositol ratio. This did not appear to affect the mutants as no growth defect was found. However, null mutations of both GAT1 and GAT2(SCT1) are synthetically lethal to yeast.

Amino Acid Sequence↗

PubMed1990

Primary stimuli of icosanoid release inhibit arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. Mechanism of action of hydrogen peroxide and methyl mercury in platelets.

Icosanoid formation in platelets depends on the concentration of free arachidonate that is mainly liberated from membrane phospholipids by phospholipase A2. The concentration of free arachidonate is also controlled by the activities of the reacylating enzymes arachidonoyl-CoA synthetase and lysophospholipid acyltransferase. In human platelet microsomes we determined the high enzyme activities of 5.9 nmol.min-1.(10(9) platelets)-1 for the arachidonoyl-CoA synthetase and 37 nmol.min-1.(10(9) platelets)-1 for the lysophospholipid acyltransferase. The activities of these reacylating enzymes were strongly reduced by hydrogen peroxide (H2O2) and methyl mercury that are primary stimuli of arachidonate release in intact platelets. H2O2 inhibited the arachidonoyl-CoA synthetase with an IC50 of 3.3 mmol/l without affecting the lysophospholipid acyltransferase. Sulfhydryl group protection by 3-mercapto-1,2-propanediol did not overcome the inhibition but glutathione prevented the inhibition of the arachidonoyl-CoA synthetase by H2O2. This suggests that glutathione by virtue of the glutathione peroxidase reduces H2O2 rather than that it protects free sulfhydryl groups of the arachidonoyl-CoA synthetase. Methyl mercury left the arachidonoyl-CoA synthetase activity unaffected but inhibited the lysophospholipid acyltransferase activity with an IC50 of 3.4 mumol/l. The inhibition is probably evoked by the blockade of sulfhydryl groups of the lysophospholipid acyltransferase because it disappeared when 3-mercapto-1,2-propanediol was added at a concentration higher than that of methyl mercury. Thrombin as a physiological full agonist, Ca2+ less than or equal to 1 mmol/l, the calcium ionophore A23187 and phorbol 12-myristate 13-acetate (TPA) and 1-oleoyl-2-acetylglycerol as model stimuli of protein kinase C neither influenced arachidonoyl-CoA synthetase nor lysophospholipid acyltransferase. It is concluded that the inhibitory effect of H2O2 and methyl mercury on the arachidonate-reacylating enzymes arachidonoyl-CoA synthetase or lysophospholipid acyltransferase, respectively, are responsible for their capacity to stimulate icosanoid release in intact cells. Thrombin and its intracellular messengers Ca2+ and diacylglycerol do not directly affect arachidonoyl-CoA synthetase and lysophospholipid acyltransferase.

Acyltransferases↗

PubMed1994

Molecular characterization of three loss-of-function mutations in the isopenicillin N-acyltransferase gene (penDE) of Penicillium chrysogenum.

Five mutants of Penicillium chrysogenum blocked in penicillin biosynthesis (npe) which are deficient in isopenicillin N-acyltransferase were isolated previously. Three of these mutants, npe6, npe7, and npe8, have been characterized at the molecular level and compared with npe10, a deletion mutant. Transcripts of normal size (1.15 kb) of the penDE genes, which encode isopenicillin N-acyltransferase, and also of the pcbAB (11.5 kb) and pcbC (1.1 kb) genes were observed in all mutants except for the npe10 mutant. Immunoblotting studies using antibodies against isopenicillin N-acyltransferase showed that all mutants (except npe10) formed the 40-kDa (unprocessed) protein and the 29-kDa subunit of the isopenicillin N-acyltransferase. The 11-kDa subunit could not be observed in the immunoblots. The mutant penDE genes of strains npe6, npe7, and npe8 were cloned and sequenced. These three strains showed a mutation in the penDE genes which results in a single amino acid change in each modified isopenicillin N-acyltransferase. The mutation in npe6 resulted in a change of Gly-150 to Val, whereas the mutation in both npe7 and npe8 introduced a change of Glu-258 to Lys. Replacement of the Val-150 and Lys-258 mutations by constructing hybrid isopenicillin N-acyltransferase molecules led to the recovery of the isopenicillin N-acyltransferase activity. The mutations in npe6, npe7, and npe8 do not affect the ability of the 40-kDa isopenicillin N-acyltransferase to be processed into the component subunits.

Acyltransferases↗

PubMed2002

Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent.

The bacterial acyltransferases of the SxxK superfamily vary enormously in sequence and function, with conservation of particular amino acid groups and all-alpha and alpha/beta folds. They occur as independent entities (free-standing polypeptides) and as modules linked to other polypeptides (protein fusions). They can be classified into three groups. The group I SxxK D,D-acyltransferases are ubiquitous in the bacterial world. They invariably bear the motifs SxxK, SxN(D), and KT(S)G. Anchored in the plasma membrane with the bulk of the polypeptide chain exposed on the outer face of it, they are implicated in the synthesis of wall peptidoglycans of the most frequently encountered (4-->3) type. They are inactivated by penicillin and other beta-lactam antibiotics acting as suicide carbonyl donors in the form of penicillin-binding proteins (PBPs). They are components of a morphogenetic apparatus which, as a whole, controls multiple parameters such as shape and size and allows the bacterial cells to enlarge and duplicate their particular pattern. Class A PBP fusions comprise a glycosyltransferase module fused to an SxxK acyltransferase of class A. Class B PBP fusions comprise a linker, i.e., protein recognition, module fused to an SxxK acyltransferase of class B. They ensure the remodeling of the (4-->3) peptidoglycans in a cell cycle-dependent manner. The free-standing PBPs hydrolyze D,D peptide bonds. The group II SxxK acyltransferases frequently have a partially modified bar code, but the SxxK motif is invariant. They react with penicillin in various ways and illustrate the great plasticity of the catalytic centers. The secreted free-standing PBPs, the serine beta-lactamases, and the penicillin sensors of several penicillin sensory transducers help the D,D-acyltransferases of group I escape penicillin action. The group III SxxK acyltransferases are indistinguishable from the PBP fusion proteins of group I in motifs and membrane topology, but they resist penicillin. They are referred to as Pen(r) protein fusions. Plausible hypotheses are put forward on the roles that the Pen(r) protein fusions, acting as L,D-acyltransferases, may play in the (3-->3) peptidoglycan-synthesizing molecular machines. Shifting the wall peptidoglycan from the (4-->3) type to the (3-->3) type could help Mycobacterium tuberculosis and Mycobacterium leprae survive by making them penicillin resistant.

Acyltransferases↗

PubMed1989

Hepatic monoacylglycerol acyltransferase: ontogeny and characterization of an activity associated with the chick embryo.

Hepatic monoacylglycerol acyltransferase is expressed during the perinatal period in rats and guinea pigs and appears to be related temporally to the availability of fatty acids and to the development of hepatic steatosis. In order to determine when monoacylglycerol acyltransferase activity is expressed in an avian species, its ontogeny was investigated in chick liver total particulate preparations. In livers from 11- to 21-day-old chick embryos, monoacylglycerol acyltransferase specific activity was 34.5 +/- 8.1 nmol/min per mg of total particulate protein. The specific activity decreased 93% to 2.6 +/- 1.3 nmol/min per mg by the 6th day after hatching. The specific activities of fatty acid CoA ligase, diacylglycerol acyltransferase, and microsomal and mitochondrial glycerol-P acyltransferases changed comparatively little during this time period. In the embryos, the monoacylglycerol acyltransferase activity per liver rose 28-fold between the 11th and 21st day, corresponding exactly to the increase in liver total particulate protein during this time. Monoacylglycerol acyltransferase activity in other tissues was 25- to 115-fold lower than observed in liver. Optimal activity was measured using 25 microM palmitoyl-CoA and 50 microM sn-2-monooleoylglycerol. The activity with the 1- and 2-monooleoylglycerol ethers and 1-monooleoylglycerol was very low. In contrast to microsomes from rat liver, about 70% of the product with the 1- and 2-monooleoylglycerol ethers was triradylglycerol, suggesting that the diacylglycerol acyltransferase from chick liver can acylate acyl, alkylglycerols. The activity with sn-2-monooleoylglycerol amide was 12.5% of that observed with the corresponding 2-monooleoylglycerol suggesting that the ester bond is important; the 1-monooleoylglycerol amide was not a substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyltransferases↗

PubMed1981

Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

The relationship between plasma lecithin:cholesterol acyltransferase mass and enzyme activity and between mass and plasma cholesterol esterification rate was determined in 25 adult volunteers without overt disease (14 normolipidemic and 11 hyperlipidemic). Furthermore, the relationship of lecithin:cholesterol acyltransferase mass and cholesterol esterification rate to lipids, apoproteins, age, and ideal body weight was assessed. Lecithin:cholesterol acyltransferase mass determined by radioimmunoassay was highly correlated with enzyme activity assayed using a heated plasma substrate (r = 0.636) and with the molar cholesterol esterification rate determined either by radioassay (r = 0.809) or by measurement of the decrease of unesterified cholesterol (r = 0.621). Lecithin:cholesterol acyltransferase mass was also positively correlated with total cholesterol (r = 0.608), unesterified cholesterol (r = 0.562), age (r = 0.544), and percent ideal body weight (r = 0.619), but was not significantly correlated with log triglyceride, high density lipoprotein cholesterol, or apolipoproteins A-I, A-II, or D. Plasma cholesterol esterification rate by both methods was highly positively correlated with total cholesterol, unesterified cholesterol, log triglyceride, and age, but was inversely correlated with high density lipoprotein cholesterol. Upon partial correlation analysis with lecithin:cholesterol acyltransferase mass kept constant the cholesterol esterification rate remained significantly positively related to total cholesterol, unesterified cholesterol, and log triglyceride and inversely related to high density lipoprotein cholesterol. Two subjects had normal lecithin:cholesterol acyltransferase but approximately half normal molar cholesterol esterification rate. Measurement of lecithin:cholesterol acyltransferase mass and activity along with plasma cholesterol esterification rate will permit differentiation of abnormalities of enzyme from qualitative or quantitative substrate or cofactor abnormalities. Also, the finding that the regression line between LCAT mass and the plasma esterification rate by direct determination of unesterified cholesterol passes through the origin suggests that all immunodetectable LCAT in plasma is active in normal subjects.-Albers, J. J., C-H. Chen, and J. L. Adolphson. Lecithin:cholesterol acyltransferase (LCAT) mass; its relationship to LCAT activity and cholesterol esterification rate.

Adult↗

PubMed1977

1-Alkyl-sn-glycero-3-phosphate: acyl-CoA acyltransferase in rat brain microsomes.

1-Alkyl-sn-glycero-3-phosphate:acyl-CoA acyltransferase activity was found in six rat tissues: heart, spleen, brain, kidney, liver, and lung. The enzyme in rat brain showed highest specific activity in the microsomal fraction and its properties were studied in detail. Triton X-100 and bovine serum albumin were found to stimulate the activity of the enzyme. It was found that Triton X-100 affects the acyltransferase activity differentially depending on the acyl-CoA donor. Albumin reversed the inhibition caused by a high concentration of 1-alkyl-sn-glycero-3-phosphate. The enzyme was shown to be stereospecific in acylating the alkylglycerophosphate substrate. Only the 1-alkyl-sn-glycero-3-phosphate isomer, not the other optical enantiomer, was active with brain acyltransferase. Evidence is presented to show that this enzyme is different from 1-acyl-sn-glycero-3-phosphate:acyl-CoA acyltransferase. Investigations of the specificity of the brain acyltransferase activity for different acyl-CoA species (16:0, 18:0, 18:2, 20:4, 22:4, 22:6) showed a selectivity which was dependent on the alkylglycerophosphate concentration. At low 1-alkyl-sn-glycero-3-phosphate concentration the enzyme selected polyunsaturated acyl-CoA species over saturated species. Providing a low but continuous amount of 1-alkyl-sn-glycero-3-phosphate to the acyltransferase was accomplished by the in situ enzymatic reduction of alkyldihydroxyacetone phosphate with NADPH in the incubation mixtures. The acyl composition of ether-containing phosphoglycerides in rat brain microsomes, as well as the free fatty acid composition, was determined. Alkenylacylglycerophosphorylethanolamine contains a high percentage of 22:6 ester, and alkenylacylglycerophosphorylcholine contains a large amount of 16:0 ester. Comparison of the acyl-CoA specificity of 1-alkyl-sn-glycero-3-phosphate acyltransferase and ether/glycerolipid acyl composition suggests that this acyltransferase could partially determine the composition of acyl groups in ether lipids.

Acetyltransferases↗

PubMed1976

Triacylglycerol synthesis in isolated fat cells. Studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerols.

The acyl-CoA:1,2-diacylglycerol acyltransferase (EC 2.3.1.20) activity of isolated fat cells was predominantly (89%) localized to the microsomal subcellular fraction by assays based on the conversion of 1,2-[3H]diacyl-sn-glycerol to triacylglycerol using 1 to 4 mug of protein. A complementary assay based on the conversion of [3H]palmitoyl-CoA to triacylglycerol was developed. These methods, 100 to 1000 times more sensitive than those previously employed, were used to characterize the microsomal activity. The choice of dispersing agent for addition of diacylglycerol to the reaction mixture was crucial. Addition of diacylglycerol in ethanol resulted in the highest diacylglycerol acyltransferase activity of the methods tested. Tween 20, which has previously been employed as the dispersing agent, severely inhibited the activity. A broad pH optimum from 7.4 to 8.0 was noted and several salts stimulated the activity more than 2-fold. The activity was unstable at temperatures of 28 degrees and above. Dependences on acyl-CoAs containing 6 to 18 carbon atoms were investigated using bacterial diacylglycerol. Acetyl- and butyryl-CoA were not substrates. Highest diacylglycerol acyltransferase activities were observed with decanoyl-CoA and lower activities were noted with longer and shorter saturated chains. Maximal activity with oleoyl-CoA was only 34% of that seen with stearoyl-CoA. No simple relationship between the critical micellar concentrations of the acyl-CoAs employed and diacylglycerol acyltransferase activity was observed. The dependences on diacylglycerols containing fatty acids 6 to 18 carbon atoms in length were investigated with [3H]palmitoyl-CoA. While all the 1,2-diacyl-sn-glycerols tested were substrates, diacylglycerol acyltransferase activity was highest with 1,2-dioleoylglycerol. Maximum activity with the bacterial diacylglycerol was 86% that with 1,2-dioleoylglycerol. The diacylglycerol concentrations required for half-maximal velocity were 20 to 40 muM for long chain diacylglycerols and 2 to 3 muM for short chain diacylglycerols; these were 75- to 750-fold lower than previously reported. Microsomal diacylglycerol acyltransferase specific activities from isolated cells around 50 nmol/min/mg, 10- to 50-fold higher than previously reported from adipose tissue, were typical using dioleoylglycerol and palmitoyl-CoA as substrates. Diacylglycerol acyltransferase specific activities were 17-fold higher in microsomes from isolated fat cells than any other tissue examined. The diacylglycerol acyltransferase appears to have specificity with respect to acyl-CoAs and diacylglycerols, but a definitive interpretation is limited by the lack of data on the physical properties of these substrates in solution under the conditions employed.

Acyltransferases↗

PubMed2005

A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters.

Acyl-CoA-dependent O-acyltransferases catalyze reactions in which fatty acyl-CoAs are joined to acyl acceptors containing free hydroxyl groups to produce neutral lipids. In this report, we characterize a human multifunctional O-acyltransferase (designated MFAT) that belongs to the acyl-CoA:diacylglycerol acyltransferase 2/acyl-CoA:monoacylglycerol acyltransferase (MGAT) gene family and is highly expressed in the skin. Membranes of insect cells and homogenates of mammalian cells overexpressing MFAT exhibited significantly increased MGAT, acyl-CoA:fatty acyl alcohol acyltransferase (wax synthase), and acyl-CoA:retinol acyltransferase (ARAT) activities, which catalyze the synthesis of diacylglycerols, wax monoesters, and retinyl esters, respectively. Furthermore, when provided with the appropriate substrates, intact mammalian cells overexpressing MFAT accumulated more waxes and retinyl esters than control cells. We conclude that MFAT is a multifunctional acyltransferase that likely plays an important role in lipid metabolism in human skin.

Acyltransferases↗

PubMed1992

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC).

An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed1984

Demonstration of intact intracellular cholesterol esterification and normal low-density lipoprotein pathway in fibroblasts from a patient with lecithin:cholesterol acyltransferase deficiency.

There was a 6-46-fold increase in intracellular cholesterol esterification in response to 25-hydroxycholesterol and low-density lipoproteins in normal and lecithin:cholesterol acyltransferase-deficient fibroblasts. Uptake and degradation of 125I-labelled low-density lipoproteins were similar in the two cell lines. Low-density lipoproteins caused a doubling of the mass of cholesteryl ester in the mutant cells. These findings indicate that: (a) acyl-CoA:cholesterol acyltransferase exhibits normal activity in mutant cells; (b) lecithin:cholesterol acyltransferase and acyl-CoA:cholesterol acyltransferase are different enzymes and are probably not products of the same gene; (c) the low-density lipoprotein-pathway is intact in fibroblasts from a patient with lecithin:cholesterol acyltransferase deficiency; (d) acyl-CoA:cholesterol acyltransferase is probably responsible for the small amount of cholesteryl ester found in plasma from patients with lecithin:cholesterol acyltransferase deficiency.

Cell Line↗

PubMed1984

The role of plasma membranes in the transfer of non-esterified cholesterol to the acyl-CoA:cholesterol acyltransferase substrate pool in liver microsomal fraction.

The incubation at 37 degrees C of rat-liver microsomal fraction followed by re-isolation of the treated microsomal vesicles results in a time-dependent increase in the activity of acyl-CoA:cholesterol acyltransferase. The rate of this increase was higher in the microsomal fraction from rats fed cholesterol-supplemented diet or starved overnight as compared with that in the microsomal fraction from rats fed standard diet. The presence of a plasma membrane preparation in the incubation mixture also resulted in a time-dependent increase in acyl-CoA:cholesterol acyltransferase activity at a rate that was dependent on the concentration of plasma membranes. During the incubation of the microsomal fraction in the presence of phosphatidylcholine liposomes, cholesterol is transferred from the microsomal to liposomal vesicles. This transfer followed first-order kinetics with respect to cholesterol concentration in the donor with a rate that increased with the concentration of liposomes in the incubation mixture. The presence of phospholipid was also associated with a decrease in the activity of the acyltransferase that was related to the concentration of phospholipid in the incubation mixture. The incubation of the microsomal fraction in the presence of phosphatidylcholine-cholesterol liposomes resulted in a time-dependent and concentration-dependent transfer of liposomal cholesterol to the microsomal fraction and the acyltransferase substrate pool. The measurement of the rate of transfer of liposomal cholesterol to the microsomal vesicles and to the acyltransferase substrate pool at various temperatures showed that activation energies for the two processes are similar. Similar to these various was also the activation energy for the increase in acyl-CoA:cholesterol acyltransferase activity due to preincubation in the absence of artificial membrane vesicles. The present results suggest that there is, under the present conditions, a time-dependent and temperature-dependent flow of cholesterol from plasma membranes to the acyltransferase substrate pool and that this flow is either diverted in the presence of phospholipid liposomes or increased in the presence of cholesterol-phospholipid liposomes.

Acyltransferases↗

PubMed1985

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred.

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.

Female↗

PubMed1987

Age-related variations in acyl-CoA:retinol acyltransferase activity and vitamin A concentration in the liver and epidermis of hairless mice.

Since the factors regulating retinol esterification by acyl-CoA:retinol acyltransferase are poorly understood, we studied the age-related variations in acyl-CoA:retinol acyltransferase activity in hairless mice. Epidermis and liver were collected at intervals from birth to adolescence (0-6 weeks). Vitamin A was analyzed by high-performance liquid chromatography and acyl-CoA:retinol acyltransferase by an in vitro radioincubation assay of microsomes. Epidermal vitamin A (retinol plus retinyl esters) increased 8-10 times after birth and by the age of 3 weeks adult values were attained. This increase was accompanied by a 2-fold increase in acyl-CoA:retinol acyltransferase activity in the epidermis between 3 days and 6 weeks of age. In young animals the dependence of acyl-CoA:retinol acyltransferase on exogenous co-substrate (palmitoyl-CoA) was also lower than in adult animals. Although a pronounced age-related accumulation of retinol was recorded in the liver, the activity of acyl-CoA:retinol acyltransferase did not increase with age and there was no change in the dependence of acyl-CoA:retinol acyltransferase on exogenous palmitoyl-CoA.

Acyltransferases↗

PubMed1989

Induction of microsomal 1-acylglycerophosphocholine acyltransferase by peroxisome proliferators in rat kidney; co-induction with peroxisomal beta-oxidation.

Induction of microsomal 1-acyl-glycerophosphocholine (GPC) acyltransferase in rat tissues by four peroxisome proliferators, clofibric acid, tiadenol, DEHP and PFOA, was examined. Among the nine tissues examined, kidney, liver and intestinal mucosa responded to the challenges by the peroxisome proliferators to induce the enzyme. The treatment of rats with various dose of clofibric acid, tiadenol, DEHP or PFOA resulted in an induction of kidney microsomal 1-acyl-GPC acyltransferase in a dose-dependent manner. Despite the structural dissimilarity of peroxisome proliferators, the induction of microsomal 1-acyl-GPC acyltransferase was highly correlated with the induction of peroxisomal beta-oxidation. The activity of microsomal 1-acyl-GPC acyltransferase was not affected by changes in hormonal (adrenalectomy, diabetes, hyperthyroidism and hypothyroidism) and nutritional (starvation, starvation-refeeding, fat-free-diet feeding and high-fat-diet feeding) states. The induction of renal microsomal 1-acyl-GPC acyltransferase was seen in mice subsequent to the administration of clofibric acid and tiadenol and in guinea pigs subsequent to the administration of tiadenol. These results may indicate that kidney microsomal 1-acyl-GPC acyltransferase is a highly specific parameter responsive to the challenges by peroxisome proliferators and may suggest that the possibility that the inductions by peroxisome proliferators of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation in kidney are co-regulated.

1-Acylglycerophosphocholine O-Acyltransferase↗

PubMed1987

1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol.

A 20,000 X g particulate preparation isolated from maturing safflower seeds catalyzed the acylation of 1-acyl-sn-glycerol 3-phosphate with acyl-CoA to form phosphatidate. The specific activity of the reaction exceeded 200 nmol min-1 mg protein-1. Although this preparation was also capable of catalyzing the acylation of sn-glycerol 3-phosphate with acyl-CoA, the hydrolysis of phosphatidate, and the acylation of 1,2-diacylglycerol, phosphatidate was the only major product when the preparation was incubated with 1-acyl-glycerol-3-P and acyl-CoA. The enzyme responsible for this phosphatidate synthesis, 1-acyl-glycerol-3-P acyltransferase, showed a strict acyl-CoA specificity. The relative order of specificity for acyl-CoA was linoleoyl = oleoyl greater than palmitoleoyl greater than elaidoyl greater than cis-vaccenoyl greater than stearoyl = palmitoyl. This observation strongly suggests that the fatty acid composition of position 2 in phosphatidate synthesized in vivo primarily depends on both the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase and the fatty acid composition of the acyl-CoA pool in the cell. Thus, the absence of saturated fatty acids at position 2 of safflower triacylglycerol may be explained in terms of the acyl-CoA specificity of the 1-acyl-glycerol-3-P acyltransferase. The fatty acid moiety esterified at position 1 of glycerol-3-P also affected the effectiveness of the reaction. The 1-acyl-glycerol-3-P acyltransferase utilized 1-acyl-glycerol-3-P molecular species in the following order of effectiveness: linoleoyl = oleoyl greater than palmitoyl. With a rise in incubation temperature, the initial rates of acylation with unsaturated acyl-CoA species increased more rapidly than those for saturated acyl-CoA species. A similar tendency was observed for saturated and unsaturated acyl acceptors. These data suggest that affinity of the acyltransferase for substrates may vary in response to changes in temperature, and that 1-acyl-glycerol-3-P acyltransferase may be involved in the alteration of the individual fatty acid compositions at positions 1 and 2 of glycerolipids in tissues grown at different temperatures. Based on these findings, further metabolism of 1-acyl-glycerol-3-P acyltransferase products could be the major factor determining the non-random distribution of fatty acids in safflower triacylglycerol.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed1984

Asymmetric reconstitution of homogeneous Escherichia coli sn-glycerol-3-phosphate acyltransferase into phospholipid vesicles.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase of Escherichia coli cytoplasmic membrane was purified in Triton X-100 (Green, P. R., Merrill, A. H., Jr., and Bell, R. M. (1981) J. Biol. Chem. 256, 11151-11159) and incorporated into mixed micelles containing Triton X-100, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, and beta-octyl glucoside. Enzyme activity was quantitatively reconstituted from the mixed micelle into single-walled phospholipid vesicles by chromatography over Sephadex G-50. Activity coeluted with vesicles of 90-nm average diameter on columns of Sepharose CL-4B and Sephacryl S-1000. These vesicles contained less than 2 Triton X-100 and 5 beta-octyl glucoside molecules/100 phospholipid molecules. Calculations suggested that up to eight 91,260-dalton glycerol-P acyltransferase polypeptides were incorporated per 90-nm vesicle. The pH dependence and apparent Km values for glycerol-P and palmitoyl-CoA of the glycerol-P acyltransferase reconstituted into vesicles were similar to those observed upon reconstitution by mixing of the enzyme in Triton X-100 with a 20-fold molar excess of sonicated phosphatidylethanolamine:phosphatidylglycerol:cardiolipin, 6:1:1. The integrity of vesicles containing glycerol-P acyltransferase was established by trapping 5,5'-dithiobis-(2-nitrobenzoic acid). Chymotrypsin inactivated greater than 95% of the glycerol-P acyltransferase in intact vesicles and cleaved the 91,260-dalton polypeptide into several vesicle-bound and several released peptides, indicating that critical domains of the enzyme are accessible in intact vesicles. Trinitrobenzene sulfonate and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene caused greater than 90% loss of glycerol-P acyltransferase in vesicles. Disruption of vesicles with Triton X-100 did not reveal significant latent activity. These data strongly suggest that the glycerol-P acyltransferase was reconstituted asymmetrically into the vesicles with its active site facing outward.

Acyltransferases↗

PubMed1983

Possible involvement of 1-acyl-glycerophosphorylinositol acyltransferase in arachidonate enrichment of phosphatidylinositol in human platelets.

Microsomes isolated from human platelets synthesize phosphatidylinositol by the action of acyl-CoA: 1-acyl-sn-glycerol-3-phosphorylinositol(1-acyl-GPI) acyltransferase. The properties of 1-acyl-GPI acyltransferase were compared with those of 1-acyl-glycerophosphorylcholine (1-acyl-GPC) acyltransferase. Apparent Km values of 1-acyl-GPI and 1-acyl-GPC acyltransferases for the corresponding acyl acceptor (lysophospholipid) were 22 and 20 microM, respectively, in the presence of arachidonoyl-CoA as fatty acyl donor. However, the Km value (1.3 microM) of 1-acyl-GPI acyltransferase for arachidonoyl-CoA was much lower than that (5.0 microM) of 1-acyl-GPC acyltransferase. Under optimal conditions, the acylation rate of 1-acyl-GPI with arachidonoyl-CoA was 2-6 times higher than with oleoyl-CoA and linoleoyl-CoA, and was very low with saturated fatty acyl-CoAs. The acylation rates with various acyl-CoAs for 1-acyl-GPI were different from those for 1-acyl-GPC. These results suggest that the reacylation pathway of 1-acyl-GPI participates in the incorporation of arachidonic acid to phosphatidylinositol in platelet microsomes. Furthermore, there were no significant effects of thrombin-activation on acyl-CoA specificity for 1-acyl-GPI and 1-acyl-GPC acyltransferase in human platelets.

Acyl Coenzyme A↗