Stereospecificity of monoacylglycerol acyltransferase activity from rat intestine and suckling rat liver

The stereospecificity of monoacylglycerol acyltransferase from rat intestinal mucosa and suckling rat liver microsomes was examined using sn-1,2-diacylglycerol kinase from Escherichia coli. With 2-monooleoyl glycerol and palmitoyl-CoA, 88 and 87.9% of the diacylglycerol synthesized by the intestinal mucosa and suckling liver, respectively, was demonstrated to be the sn-1,2-isomer. Analysis of similar preparations of these diacylglycerol products by gas-liquid chromatography-mass spectrometry indicated that most of the remaining diacylglycerol was the 1,3-isomer that probably arose via acyl-migration. These results indicate that monoacylglycerol acyltransferase is stereospecific. Measurement of acyltransferase activities in microsomes using 1- and 2-monoacyl- and monoalkylglycerols as substrates indicated that the monoacylglycerol acyltransferases from suckling liver and intestinal mucosa have different substrate specificities.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

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)     

Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacyglycerol acyltransferase activity

Microsomal monoacyglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycerol from sn-2-monoacylglycerol and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fao rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [14C]acetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [14C]oleate into triacyglycerol about 1.7-fold within 2 h, but had little effect on [14C]oleate incorporation into phospholipid. These hormonally responsive rat-hepatoma/hepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway.     

Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.     

Microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta.

The placenta plays a major role in transporting lipid to the developing foetus. Since previous studies have suggested that placental lipid transport involves intermediate esterification steps, we investigated selected microsomal and lysosomal enzymes of triacylglycerol metabolism in rat placenta. Between gestational days 10 and 14, microsomal phosphatidic acid phosphatase specific activity was 6-fold greater than the activity in adult rat liver. Phosphatidic acid phosphatase activity decreased 50% on day 15. Studies employing several different phosphorylated substrates indicated a high degree of substrate specificity. Lysosomal triacylglycerol lipase and cholesterol esterase activities decreased about 50% between days 15 and 18, then rose late in gestation. No changes were observed in the specific activities of fatty acid: CoA ligase, glycerolphosphate acyltransferase, lysophosphatidate acyltransferase, diacylglycerol acyltransferase or diacylglycerol cholinephosphotransferase during the final 12 days of gestation. Kinetic observations (competitive inhibition by alternative substrates, pH-dependence and thermal inactivation) were consistent with the hypothesis that glycerol phosphate and dihydroxyacetone phosphate can be acylated by a single microsomal enzyme in placenta. Except for fatty acid: CoA ligase, the activities of microsomal and lysosomal enzymes of triacylglycerol metabolism were comparable with those in adult rat liver. These observations are consistent with physiological studies suggesting that triacylglycerol synthetic and degradative pathways are very active in rat placenta.     

A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Acyl specificity in triglyceride synthesis by lactating rat mammary gland.

We have studied the specificity of the acyl-CoA:diglyceride acyltransferase reaction in lactating rat mammary gland to provide a rational explanation at the enzyme level for the nonrandom distribution of fatty acids in milk fat triglycerides. Acyl-CoA:diglyceride acyltransferase activity was measured using various diglyceride and radioactive acyl-CoA substrates; products were identified as triglycerides by thin-layer and gas-liquid chromatography. Most of the enzymatic activity was located in the microsomal fraction and showed a broad specificity for the acyl donors tested C10, C12, C14, C16, C18, and C18:1 CoA esters). The acyltransferase activity was highly specific for sn-1,2-diglyceride enantiomers; rac-1,3- and sn-2,3-diglycerides were relatively inactive. The acyl-CoA specificity was not affected by the type of 1,2-diglyceride acceptor offered, although dilaurin was the best acceptor and sn-1,2-dilaurin greater than sn-1,2-dimyristin greater than sn-1,2-dipalmitin greater than sn-1,2-distearin. We have previously shown that in the microsomal fraction from lactating rat mammary gland, the acyltransferase activities concerned with the conversion of sn-glycero-3-phosphate to diacylglycerophosphate show a very marked specificity for long chain acyl-CoA's. Therefore, we conclude that the predominant localization of long chain fatty acids in the 1 and 2 positions, and of shorter chain fatty acids in the 3 position of the glycerol backbone, results at least in part from the specificities of the mammary gland acyltransferases.     

Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase.

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.     

Topographic distribution of enzymes involved in glycerolipid synthesis in rat small, intestinal epithelium.

1. The enzymes involved in glycerolphosphate and monoacylglycerol acylation of rat small intestine were more active in villi than in crypts. Monoglyceride acyltransferase (EC 2.3.1.22) was found to be absent from crypts. 2. In the villi, the enzymes are mainly localized in microsomes, although low activities of palmitoyl-CoA synthetase (EC 6.2.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15) and cholinephosphotransferase (EC 2.7.8.2) are found in mitochondria. Mitochondria lack monoglyceride acyltransferase and lysolecithin acyltransferase (EC 2.3.1.23), both of which are involved in the reacylation of alimentary partial glycerides. Therefore, this process is confined to microsomes. 3. The monoacylglycerol and lysolecithin acyltransferases, as well as choline-phosphotransferase, are probably localized within the endoplasmic reticulum, since these enzymes are relatively Nagerse resistant (subtilisin; EC 3.4.2.1, compared with palmitoyl-CoA synthetase and glycerolphosphate acyltransferase, which are highly Nagarse-sensitive and therefore probably localized on the outside of the microsomes (and mitochondria). 4. The physical separation of alimentary product reacylation from de novo synthetic processes provides the basis of metabolic compartmentation observed by other workers. 5. The use of sucrose instead of a salt medium for the isolation and homogenization of small intestinal epithelial cells allowed the separation of mitochondria and microsomes by differential centrifugation without mutual contamination. 6. Phospholipids were found to stimulate glycerolphosphate acylation in vitro. 7. The glycerolphosphate and monoacylglycerol acylation pathways are not competitive.     

Biochemistry of development in insects. Stereospecific incorporation of fatty acids into triacylglycerols.

1. Previous experiments showed that fatty acids were incorporated into triacylglycerols by homogenates of Ceratitis capitata larvae far more efficiently than by pharate adult homogenates. This metabolic behaviour of both stages of development of the insect has been interpreted throughout the existence of a different acyltransferase activity. To obtain new data on the acyltransferase mechanism, a time-course of the stereospecific incorporation of labelled myristic, palmitic, oleic and linoleic acids into the sn-positions of triacylglycerols has been followed. 2. Studies on the stereospecific incorporation of labelled fatty acids confirmed previous results. Palmitic acid was mainly incorporated into sn-1 and sn-3 positions whereas position 2 exhibited a low incorporation. Myristic acid acylated sn-3 position at a higher rate than it acylated the other sn-positions. Oleic acid was more specifically distributed than palmitic acid and linoleic acid was more efficiently incorporated than the monounsaturated acid. All these data reflect substrate differences in the acyltransferase activity of larval homogenates. Pharate adult homogenates incorporated fatty acids very scarcely and mainly into positions (1 + 3). 3. Kinetics of incorporation of labelled fatty acids into the sn-positions points to a non-random distribution with respect to the major saturated and unsaturated fatty acids in triacylglycerols of larvae of Ceratitis capitata.     

Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.5), hydrolysis and transacylation, are inactivated by diethylpyrocarbonate. The reaction follows pseudo-first-order kinetics, and second-order rate constants of 1.17 mM-1min-1 for hydrolysis and 0.56 mM-1 min-1 for transacylation were obtained at pH 6.5 and 37 degrees C. The rate of inactivation is dependent on pH, showing the involvement of a group with a pK of 6.5. The difference spectra showed an increase in absorbance at 242 nm, indicating the modification of histidine residues. The activity lost by diethylpyrocarbonate modification can be partially recovered by hydroxylamine treatment. The statistical analysis of residual fractional activity versus the number of modified histidine residues leads to the conclusion that two histidine residues are essential for the hydrolytic activity, whereas transacylation activity depends on only one essential histidine. The substrate and substrate analogs protected the enzyme against inactivation by diethylpyrocarbonate, suggesting that the essential residues are located at or near the active site of the enzyme.     

Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig.

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.     

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.)

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.     

The wax ester synthase/acyl coenzyme A:diacylglycerol acyltransferase from Acinetobacter sp. strain ADP1: characterization of a novel type of acyltransferase

The wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) catalyzes the final steps in triacylglycerol (TAG) and wax ester (WE) biosynthesis in the gram-negative bacterium Acinetobacter sp. strain ADP1. It constitutes a novel class of acyltransferases which is fundamentally different from acyltransferases involved in TAG and WE synthesis in eukaryotes. The enzyme was purified by a three-step purification protocol to apparent homogeneity from the soluble fraction of recombinant Escherichia coli Rosetta (DE3)pLysS (pET23a::atfA). Purified WS/DGAT revealed a remarkably low substrate specificity, accepting a broad range of various substances as alternative acceptor molecules. Besides having DGAT and WS activity, the enzyme possesses acyl-CoA:monoacylglycerol acyltransferase (MGAT) activity. The sn-1 and sn-3 positions of acylglycerols are accepted with higher specificity than the sn-2 position. Linear alcohols ranging from ethanol to triacontanol are efficiently acylated by the enzyme, which exhibits highest specificities towards medium-chain-length alcohols. The acylation of cyclic and aromatic alcohols, such as cyclohexanol or phenylethanol, further underlines the unspecific character of this enzyme. The broad range of possible substrates may lead to biotechnological production of interesting wax ester derivatives. Determination of the native molecular weight revealed organization as a homodimer. The large number of WS/DGAT-homologous genes identified in pathogenic mycobacteria and their possible importance for the pathogenesis and latency of these bacteria makes the purified WS/DGAT from Acinetobacter sp. strain ADP1 a valuable model for studying this group of proteins in pathogenic mycobacteria.     

SLC1 and SLC4 encode partially redundant acyl-coenzyme A 1-acylglycerol-3-phosphate O-acyltransferases of budding yeast

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1Delta cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1Delta and slc4Delta cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1Delta and slc4Delta cells incubated with [14C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1Delta and slc4Delta cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg2+-dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Localization of intracellular triacylglycerol and cholesteryl ester transfer activity in rat tissues

A triacylglycerol and cholesteryl ester transfer activity has been isolated from rat liver. After homogenization, the liver cells were subfractionated into the 10 000 X g pellet, microsomal fraction and postmicrosomal supernatant. Most of the transfer activity appeared to be associated with the microsomal fraction. Rough and smooth microsomes contained nearly equal transfer activities. When isolated microsomes were subject to proteolytic attack, the transfer activity was not inactivated, unless it had been released from the microsomes prior to proteolytic treatment. This indicates that the activity is probably located within the microsomal vesicles. Similar transfer activities were found in the intestinal mucosa of rats, whereas little or no activity was detected in the brain, heart, kidney, or plasma.