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.     

Selective acyl transfer in the reacylation of brain glycerophospholipids. Comparison of three acylation systems for 1-alk-1'-enylglycero-3-phosphoethanolamine, 1-acylglycero-3-phosphoethanolamine and 1-acylglycero-3-phosphocholine in rat brain microsomes

The activities of three acylation systems for 1-alkenylglycerophosphoethanolamine (1-alkenyl-GPE), 1-acyl-GPE and 1-acylglycerophosphocholine (1-acyl-GPC) were compared in rat brain microsomes and the acyl selectivity of each system was clarified. The rate of CoA-independent transacylation of 1-[3H]alkenyl-GPE (approx. 4.5 nmol/10 min per mg protein) was about twice as high as in the case of 1-[3H]acyl-GPE and 1-[14C]acyl-GPC. On the other hand, the rates of CoA-dependent transacylation and CoA + ATP-dependent acylation (acylation of free fatty acids by acyl-CoA synthetase and acyl-CoA acyltransferase) of lysophospholipids were in the order 1-acyl-GPC greater than 1-acyl-GPE much greater than 1-alkenyl-GPE. HPLC analysis of newly synthesized molecular species revealed that the CoA-independent transacylation system exclusively esterified docosahexaenoate and arachidonate, regardless of the lysophospholipid class. The CoA-dependent transacylation and CoA + ATP-dependent acylation systems were almost the same with respect to the selectivities for unsaturated fatty acids when the same acceptor lysophospholipid was used, but some distinctive acyl selectivities were observed with different acceptor lysophospholipids. 1-Alkenyl-GPE selectively acquired only oleate in these two systems. 1-Acyl-GPE and 1-acyl-GPC showed selectivities for both arachidonate and oleate. In addition, an appreciable amount of palmitate was transferred to 1-acyl-GPC, not to 1-acyl-GPE, in CoA- or CoA + ATP-dependent manner. The acylation of exogenously added acyl-CoA revealed that the acyl selectivities of the CoA-dependent transacylation and CoA + ATP-dependent acylation systems may be mainly governed through the selective action of acyl-CoA acyltransferase. The preferential utilization of oleoyl-CoA by all acceptors and the different utilization of arachidonoyl-CoA between alkenyl and acyllysophospholipids indicated that there might be two distinct acyl-CoA:lysophospholipid acyltransferases that discriminate between oleoyl-CoA and arachidonoyl-CoA, respectively. Our present results clearly show that all three microsomal acylation systems can be active in the reacylation of three major brain glycerophospholipids and that the higher contribution of the CoA-independent system in the reacylation of ethanolamine glycerophospholipids, especially alkenylacyl-GPE, may tend to enrich docosahexaenoate in these phospholipids, as compared with in the case of diacyl-GPC.     

Synthesis of 1-palmitoyl and 1-stearoyl phosphatidylcholines from mixtures of acyl acceptors via acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in liver microsomes.

The fatty acid selectivity of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat liver microsomes was studied using a mixture of the [1-(3)H]palmitoyl plus [1-(14C)stearoyl molecular species of 1-acylglyceryl-phosphorylcholine. At a 1-acyl-sn-glycero-3-phosphorylcholine concentration of 0.16 mM, the enzyme exhibited a selectivity of 3.5-fold for the 1-palmitoyl over the 1-stearoyl species of the acyl acceptor and reaction velocities with linoleoyl- and arachidonoyl-CoA were 38--47% greater than with oleoyl-CoA. Lowering the acceptor concentration to 0.016 mM gave reaction rates with the polyenoic thiolesters which were 174--187% greater than with oleoyl-CoA and the 1-palmitoyl-sn-glycero-3-phosphorylcholine was preferred by 2.2, 1.6, and 1.6-fold with oleoyl-, linoleoyl- and arachidonoyl-CoA, respectively. The results support the potential importance of the fatty acid selectivities of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase towards both acyl acceptor and donor in regulating the phosphatidylcholine species formed by the reaction in vivo.     

Phospholipid acylation by mouse sciatic nerve microsomes.

The partition of 0.3 nmol of [1-14C]oleoyl-CoA in the microsomes (10 micrograms proteins) from mouse sciatic nerves is unaffected by the presence of lysophospholipids and is about 45% of the total oleoyl-CoA (77% of the acylglycerophosphocholine partition in the membrane). The concentration of both oleoyl-CoA and acylglycerophosphocholine is over 1 mM in the membrane. There is a selective acyl transfer from acyl-CoA to lysolipid acceptors (oleoyl greater than myristoyl, palmitoyl, stearoyl much greater than eicosanoyl greater than docosanoyl, tetracosanoyl). The exogenous acyl acceptors are acylglycerophosphocholine and acylglycerophosphoinositol and to a lesser extent acylglycerophosphoethanolamine, but not acylglycerophosphoserine. A PC formation from acylGPC in the absence of exogenous acyl donors or from oleoyl-CoA in the absence of exogenous acyl acceptor was also observed.     

Acylation of 1-alkenyl-glycerophosphocholine and 1-acyl-glycerophosphocholine in guinea pig heart.

The deacylation-reacylation process has been shown to be an important pathway for phospholipids to attain the desired acyl groups at the C-2 position. The acylation of 1-acyl-glycerophosphocholine (-GPC) in mammalian hearts has been well documented, but the acylation of 1-alkenyl-GPC has not been described. In this paper, we demonstrate the presence of acyl-CoA: 1-alkenyl-GPC acyltransferase for the acylation of 1-alkenyl-GPC in mammalian hearts; the highest activity is found in guinea pig heart. The guinea pig heart 1-alkenyl-GPC acyltransferase has only 10-40% of the 1-acyl-GPC acyltransferase activity, and both activities are located in the microsomal fraction. However, these two enzymes respond differently to cations, detergents and heat treatment, and the two enzymes also display different acyl specificity. Kinetic studies indicate that both reactions could not be accommodated by the same catalytic site. The results provide strong evidence that the two activities are from separate and distinct proteins. The specificity of 1-alkenyl-GPC acyltransferase for unsaturated species of acyl-CoA may play an important role in the maintenance of the high degree of unsaturated acyl groups found in guinea pig heart plasmalogens.     

Glycerol 3-phosphate acylation in microsomes of type II cells isolated from adult rat lung

Glycerol 3-phosphate acylation was studied in type II cells isolated from adult rat lung. The process was found to be largely microsomal. In the microsomes phosphatidic acid is the main product of glycerol 3-phosphate acylation. Glycerol-3-phosphate acyltransferase is rate limiting in the phosphatidic acid formation by the microsomes. Type II cell microsomes incorporate palmitoyl and oleoyl residues into phosphatidic acid at an equal rate if palmitoyl-CoA and oleoyl-CoA are added separately. However, if palmitoyl-CoA and oleoyl-CoA are added as an equimolar mixture the unsaturated fatty acyl moiety is incorporated much faster. Under the latter conditions monoenoic species constitute the most abundant products of glycerol 3-phosphate acylation. The microsomes incorporate both palmitoyl and oleoyl residues readily into both the 1- and 2-position of phosphatidic acid, even when palmitoyl-CoA and oleoyl-CoA are added together. Assuming that both phosphatidic acid phosphatase and cholinephosphotransferase do not discriminate against substrates with an unsaturated acyl moiety at the 1-position and a saturated acyl moiety at the 2-position, the last two observations indicate that a considerable percentage of phosphatidylcholine molecules synthesized de novo may have a saturated fatty acid at the 2-position and an unsaturated fatty acid at the 1-position, and that remodeling at the 1-position may be important for the formation of surfactant dipalmitoylphosphatidylcholine. They also indicate that type II cell microsomes are capable of synthesizing the dipalmitoyl species of phosphatidic acid. However, since there is a preference for the acylation of glycerol 3-phosphate with unsaturated fatty acyl residues, the percentage of dipalmitoyl species in the synthesized phosphatidic acid, and thereby the percentage of dipalmitoyl species in the phosphatidylcholine synthesized de novo, will probably depend on the relative availability of the various acyl-CoA species.     

The biosynthesis of phosphatidylserines by acylation of 1-acyl-sn-glycero-3-phosphoserine in rat liver

The conversion of 1-[14C]acyl-sn-glycero-3-phosphoserine into molecular species of [14C]phosphatidylserine was studied using rat liver homogenate and microsomal preparations in the absence of added fatty acyl moieties. In liver homogenates, 81% of the newly-formed phosphatidylserines were tetraenoic (arachidonoyl) species while saturated, monoenoic, dienoic, trienoic, pentaenoic, and hexaenoic (docosahexaenoyl) species each represented 2-5% of the total. A similar pattern of molecular species was produced in liver microsomes. The selectivity of the microsomal acyl-CoA:1-acyl-sn-glycero-3-phosphoserine acyltransferase towards different acyl-CoA derivatives was also investigated. The relative suitability of the various acyl-CoA esters as substrates was found to be of the following order:20:4 = 18:2 greater than 18:1 greater than 16:0 = 18:0. These results with endogenous acyl donors suggest that the acylation of 1-acyl-sn-glycero-3-phosphoserine may partly account for the enrichment of liver phosphatidylserine in arachidonic acid but does not appear to be primarily responsible for the preponderance of docosahexaenoic acid in this phospholipid. The fatty acid specificity of the acyl-CoA: 1-acyl-sn-glycero-3-phosphoserine acyltransferase may contribute to the preferential formation of arachidonoyl phosphatidylserine.     

ACYLATION OF LYSOPHOSPHATIDYLSERINE BY RAT BRAIN MICROSOMES

Abstract— The acylation of lysophosphatidylserine, prepared by snake venom digestion of phosphatidylserine, by rat brain microsomes is described. Acylation was monitored by spectrophotometric assay and by measuring the incorporation of radioactively labelled acyl CoA thioesters. Acylation was time dependent, showed an approximately linear response to enzyme concentration and had a pH optimum of 9.0. Maximum acylation was attained at a concentration of about 100 μM for lysophosphatidylserine and about 40μM for acyl CoA thioesters. Positional distribution studies with [¹⁴C]oleoyl CoA and [¹⁴C]arachidonoyl CoA showed incorporation was predominantly at position -2, but with significant labelling at position–1, particularly with oleoyl CoA, possibly as a result of isomerization of the 1–acyl isomer of lysophosphatidylserine. Both saturated and unsaturated thioesters could serve as acyl group donors. Myristoyl CoA was considerably superior to palmitoyl CoA and stearoyl CoA, which were poor acyl group donors. Some selectivity was shown among the long chain unsaturated thioesters, linoleoyl, linolenoyl and arachidonoyl CoA being the most effective acylating agents. Although docosahexaenoic acid is a major unsaturated fatty acid in brain phosphatidylserine, its CoA ester was a relatively poor acyl group donor. Relative acylation rates remained essentially constant over a wide range of lysophosphatidylserine concentrations. It is concluded that acyl transfer mechanisms are active in brain for the regulation of the fatty acid profile of phosphatidylserine.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

Substrate selectivity of plant and microbial lysophosphatidic acid acyltransferases

Linoleic acid (18:2) is found in a large variety of plant oils but to date there is limited knowledge about the substrate selectivity of acyltransferases required for its incorporation into storage triacylglycerols. We have compared the incorporation of oleoyl (18:1) and linoleoyl (18:2) acyl-CoAs onto lysophosphatidic acid acceptors by sub-cellular fractions prepared from a variety of plant and microbial species. Our assays demonstrated: (1). All lysophosphatidic acid acyltransferase (LPA-AT) enzymes tested incorporated 18:2 acyl groups when presented with an equimolar mix of 18:1 and 18:2 acyl-CoA substrates. The ratio of 18:1 to 18:2 incorporation into phosphatidic acid varied between 0.4 and 1.4, indicating low selectivity between these substrates. (2). The presence of either stearoyl (18:0) or oleoyl (18:1) groups at the sn-1 position of lysophosphatidic acid did not affect the selectivity of incorporation of 18:1 or 18:2 into the sn-2 position of phosphatidic acid. (3). All LPA-AT enzymes tested incorporated the saturated palmitoyl (16:0) acyl group from equimolar mixtures of 16:0- and 18:1-CoA. The ratios of 18:1 to 16:0 incorporation are generally much higher than those of 18:1 to 18:2 incorporation, varying between 2.1 and 8.6. (4). The LPA-AT from oil palm kernel is an exception as 18:1 and 16:0 are utilised at comparable rates. These results show that, in the majority of species examined, there is no correlation between the final sn-2 composition of oil or membrane lipids and the ability of an LPA-AT to use 18:2 as a substrate in in vitro assays.     

Selective acylation of alkyllysophospholipids by docosahexaenoic acid in Ehrlich ascites cells

Ehrlich ascites cells were cultured with 1-O-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) or 1-O-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC) to reveal the selective retention of polyunsaturated fatty acids at second position of ether-containing phospholipids. Although small percentages of the lysophospholipids were degraded into long-chain alcohol, both alkyllyso-GPE and -GPC were acylated at the rate of approximately 2 nmol/30 min per 10(7) cells. Alkylacylacetylglycerols were prepared from the acylated products by phospholipase C treatment, acetylation and TLC, and fractionated according to the degree of unsaturation by AgNO3-TLC. The distribution of the radioactivity among the subfractions indicated that both alkyllysophospholipids were mainly esterified by docosahexaenoic acid and to a somewhat lesser extent by arachidonic acid. The selectivity for docosahexaenoic acid in the esterification of 1-alkyl-GPE was much stronger than in that of 1-alkyl-GPC. Although acyl-CoA: 1-alkyl-glycerophosphoethanolamine acyltransferase activity of Ehrlich cell microsomes with arachidonoyl-CoA and docosahexaenoyl-CoA as acyl donors was negligible compared with the acyl-CoA:1-alkyl-glycerophosphocholine acyltransferase activity, a significant amount of 1-alkyl-GPE was acylated in the microsomes without exogenously added acyl-CoA. HPLC analysis revealed that docosahexaenoic acid and arachidonic acid were mainly esterified by the microsomal transferase. Acylation of 1-alkyl-GPC with docosahexaenoic acid and arachidonic acid was also observed in the absence of added acyl-CoA, but the activity was lower than that for 1-alkyl-GPE. Although the source of the acyl donor in the acylation has not been determined, the acylation is probably due to the direct transfer of acyl groups between intact phospholipids. The above results provided the first evidence that the lysophospholipid acyltransferase system including the transacylase activity participates in the selective retention of docosahexaenoic acid in intact cells and a cell free system.     

The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds

Microsomes isolated from the developing cotyledons of the seeds of the safflower varieties, very-high-linoleate, Gila and high-oleate, were capable of exchanging the acyl groups in acyl-CoA with the fatty acids in position 2 of phosphatidylcholine. The specificity of the 'acyl-exchange' towards the acyl moiety in acyl-CoA was selective in the order: oleate greater than linoleate greater than linolenate. Stearoyl-CoA was completely selected against when presented in a mixed substrate with unsaturated 18-carbon acyl-CoAs. Microsomes, of the very-high-linoleate safflower variety, rapidly desaturated in situ-labelled [14C]oleoylphosphatidylcholine in the presence of NADH. Little oleate desaturation, however, was observed in the microsomes of the high-oleate variety. Microsomes of the Gila and high-oleate varieties of safflower rapidly synthesised phosphatidic acid by the acylation of glycerol 3-phosphate with acyl-CoA. The phosphatidic acid was metabolised to diacylglycerol, which was further acylated to triacylglycerol. A strong selectivity for linoleoyl-CoA was found for the acylation of glycerol 3-phosphate in both the Gila and high-oleate microsomes. On the basis of these results, we propose that the pattern of 18-carbon unsaturated fatty acids in the triacylglycerols of all 'oil'-producing seeds is a direct reflection of the fatty acids in the acyl-CoA pool. This, in turn, is governed by: A, the rate and specificity of the acyl exchange between acyl-CoA and phosphatidylcholine; B, the rate of oleate (and linoleate) desaturation in phosphatidylcholine; and C, the rate and specificity of the glycerophosphate acyltransferase.     

Studies on lipid metabolism in Tetrahymena pyriformis: properties of acyltransferase systems.

Membrane preparations from Tetrahymena pyriformis catalyzed the acylations of glycerophosphate, isomeric monoacylglycerophosphate, and 1-acylglycerylphosphoryl-choline. Under the optimal conditions, glycerophosphate acyltransferase and 1-acylgly-cerophosphate acyltransferase used saturated and unsaturated acyl-CoA at comparable rates. The specificities of these acyltransferase systems for various acyl-CoAs as compared with the respective maximal velocities do not directly explain the fatty acid distribution in glycerophospholipids. However, the acylation of 2-acylglycerophosphate was highly selective for palmitate when the incubations were carried out in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The 1-acylglycerylphosphorylcholine acyltransferase system showed relatively higher specificity for unsaturated acyl-CoA, which is consistent with the fatty acid pattern of phospholipids. Significant amounts of diglyceride and triglyceride were formed together with phosphatidic acid from acyl-CoA and glycerophosphate, indicating that the enzymes involved in triglyceride synthesis are closely associated with acyltransferase systems involved in phosphatidate synthesis in microsomes. These acyltransferase activities were found mainly in microsomes, and to a lesser extent, in pellicles, too. No significant difference was observed in the properties of acyltransferase systems in microsomes and pellicles.     

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C₁₈-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.     

Oleate metabolism in microsomes from developing leaves ofPisum sativum L.

Microsomes from young leaves of pea,Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA:lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoyl-CoA:phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties.Abbreviations FA unesterified fatty acid (s) - PC phosphatidylcholines - 18:1 oleoyl moieties - 18:2 lmoleoyl moieties Dedicated to Professor Helmut K. Mangold, Bundesanstalt für Fettforschung, Münster, on his 60th birthday     

Solubilization and modulation of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase activity in rat liver microsomes

The acylation of 1-acyl-glycerophosphocholine is an important mechanism for the maintenance of the asymmetrical distribution of acyl groups in phosphatidylcholine. The majority of acyl-CoA:1-acyl-glycerophosphocholine acyltransferase is located in the microsomal fraction. In this study, the rat liver microsomes were incubated with various detergents, and the solubilized enzyme was separated from the remainder by centrifugation. Sodium cholate, sodium deoxycholate and octylglucopyranoside caused the Solubilization of 14–25% of the enzyme activity. The acyl specificity of the solubilized enzyme was similar to the insoluble enzyme, indicating that there was no selective solubilization of any acyl specific acyltransferase. The solubilized enzyme did not display any lipid requirement, and its activity was inhibited by phosphatidylcholine, phosphatidylethanolamine and 1,2-diacylglycerol. Kinetic studies with varying concentrations of acyl-CoAs revealed that the inhibition by 1,2-diacylglycerol was essentially uncompetitive. The modulation of acyltransferase activity by 1,2-diacylglycerol may be an important mechanism for controlling the acylation of lysophosphatidylcholine.     

Purification and kinetic properties of lysophosphatidylinositol acyltransferase from bovine heart muscle microsomes and comparison with lysophosphatidylcholine acyltransferase

The enzyme acyl-CoA:1-acyl-sn-glycero-3-phosphoinositol acyltransferase (LPI acyltransferase, EC 2.3.1.23) was purified approximately 11,000-fold to near homogeneity from bovine heart muscle microsomes. The purification was effected by extraction with the detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate, followed by chromatography on Cibacron blue agarose, DEAE-cellulose, and Matrex gel green A. The isolated enzyme was a single protein of 58,000 Da as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. This purification procedure also allows isolation of the related enzyme lysophosphatidylcholine (LPC) acyltransferase, which was separated from LPI acyltransferase at the final chromatographic step. The purified LPI acyltransferase exhibits an absolute specificity for LPI as the acyl acceptor. Broader specificity was found for acyl-CoA derivatives as substrates, although the preferred substrates are long-chain, unsaturated derivatives: measured reactivities were in the order arachidonoyl-CoA greater than oleoyl-CoA greater than eicosadienoyl-CoA greater than linoleoyl-CoA. Little activity was found with palmitoyl-CoA or stearoyl-CoA as potential substrates. These properties are consistent with a role of the enzyme in controlling the acyl group composition of phosphoinositides. Comparison of LPC acyltransferase and LPI acyltransferase shows that these two enzymes have distinct kinetic and physical properties and are affected differently by local anesthetics, which are potent inhibitors.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Acyl coenzyme A: cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at −40°C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

Reverse reaction of lysophosphatidylinositol acyltransferase. Functional reconstitution of coenzyme A-dependent transacylation system

CoA-dependent transacylation activity in microsomes catalyzes the transfer of fatty acid between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acid. We examined the mechanism of the transacylation system using partially purified acyl-CoA:lysophosphatidylinositol (LPI) acyltransferase (LPIAT) from rat liver microsomes to test our hypothesis that both the reverse and forward reactions of acyl-CoA:lysophospholipid acyltransferases are involved in the CoA-dependent transacylation process. The purified LPIAT fraction exhibited ATP-independent acyl-CoA synthetic activity and CoA-dependent LPI generation from PI, suggesting that LPIAT could operate in reverse to form acyl-CoA and LPI. CoA-dependent acylation of LPI by the purified LPIAT fraction required PI as the acyl donor. In addition, the combination of purified LPIAT and recombinant lysophosphatidic acid acyltransferase could reconstitute CoA-dependent transacylation between PI and phosphatidic acid. These results suggest that the CoA-dependent transacylation system consists of the following: 1) acyl-CoA synthesis from phospholipid through the reverse action of acyl-CoA:lysophospholipid acyltransferases; and 2) transfer of fatty acyl moiety from the newly formed acyl-CoA to lysophospholipid through the forward action of acyl-CoA:lysophospholipid acyltransferases.