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.     

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.     

Effects of substrate fatty acids on products of lecithin hydrolysis and acyl-CoA-independent transacylation with cholesterol by aortic enzyme preparations

The acyl composition of substrates and products of enzymatic hydrolysis and transacylation of lecithin with cholesterol in the arterial wall was investigated. Saturated acyl residues predominated in lysolecithin and unsaturated ones in acids released by hydrolysis of egg lecithin. In the reaction system with cholesterol, saturated acyls predominated in both lysolecithin and acids released whereas unsaturated ones were more abundant in newly formed acylcholesterols. Mainly unsaturated acyls were present in the hydrolysis products from soybean lecithin in the reaction systems with and without cholesterol. For acylcholesterols formed in the presence of either lecithin, the percent values are in the numerical order of C18:2 greater than C18:1 greater than C16:0 greater than or equal to C18:0. It It is concluded that acyl preferences and interactions in the enzyme-catalyzed reactions studied may contribute to the different accumulation and removal of the compounds involved from the artery.     

The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase

The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6- amino acid substitution into the putative active site (amino acids 249-254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464-523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.     

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.     

ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.     

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.     

Characterization of an acyl-coenzyme a thioesterase associated with the envelope of spinach chloroplasts

The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell.     

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.     

Characterization of the Acyl-CoA synthetase activity of purified murine fatty acid transport protein 1

Fatty acid transport protein 1 (FATP1) is an approximately 63-kDa plasma membrane protein that facilitates the influx of fatty acids into adipocytes as well as skeletal and cardiac myocytes. Previous studies with FATP1 expressed in COS1 cell extracts suggested that FATP1 exhibits very long chain acyl-CoA synthetase (ACS) activity and that such activity may be linked to fatty acid transport. To address the enzymatic activity of the isolated protein, murine FATP1 and ACS1 were engineered to contain a C-terminal Myc-His tag expressed in COS1 cells via adenoviral-mediated infection and purified to homogeneity using nickel affinity chromatography. Kinetic analysis of the purified enzymes was carried out for long chain palmitic acid (C16:0) and very long chain lignoceric acid (C24:0) as well as for ATP and CoA. FATP1 exhibited similar substrate specificity for fatty acids 16-24 carbons in length, whereas ACS1 was 10-fold more active on long chain fatty acids relative to very long chain fatty acids. The very long chain acyl-CoA synthetase activity of the two enzymes was comparable as were the Km values for both ATP and coenzyme A. Interestingly, FATP1 was insensitive to inhibition by triacsin C, whereas ACS1 was inhibited by micromolar concentrations of the compound. These data represent the first characterization of purified FATP1 and indicate that the enzyme is a broad substrate specificity acyl-CoA synthetase. These findings are consistent with the hypothesis that that fatty acid uptake into cells is linked to their esterification with coenzyme A.     

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.     

Involvement of acyl exchange between acyl-CoA and phosphatidylcholine in the remodelling of phosphatidylcholine in microsomal preparations of rat lung

Microsomal membrane preparations from rat lung catalyse the incorporation of radioactive linolenic acid from [14C]linolenoyl-CoA into position 2 of sn-phosphatidylcholine. The incorporation was stimulated by bovine serum albumin and free CoA. Free fatty acids in the incubation mixtures were not utilised in the incorporation into complex lipids. Fatty acids were transferred to the acyl-CoA pool during the incorporation of linolenic acid into phosphatidylcholine. An increase in lysophosphatidylcholine occurred in incubations containing both bovine serum albumin and free CoA and in the absence of acyl-CoA. The results were consistent with an acyl-CoA: lysophosphatidylcholine acyltransferase operating in both a forwards and backwards direction and thus catalysing the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine. In incubations with mixed species of acyl-CoAs, palmitic acid was the major fatty acid substrate transferred to phosphatidylcholine in acyl exchange, whereas this acid was completely selected against in the acylation of added lysophosphatidylcholine. The selectivity for palmitoyl-CoA was particularly enhanced when the mixed acyl-CoA substrate was presented to the microsomes in molar concentrations equivalent to the molar ratios of the fatty acids in position 2 of sn-phosphatidylcholine. During acyl exchange, the predominant fatty acid transferred to phosphatidylcholine from acyl-CoA was palmitic acid, whereas arachidonic acid was particularly selected for in the reverse reaction from phosphatidylcholine to acyl-CoA. A hypothesis is presented to explain the differential selectivity for acyl species between the forward and backward reactions of the acyltransferase that is based upon different affinities of the enzyme for substrates at high and low concentrations of acyl donor. Acyl exchange between acyl-CoA and phosphatidylcholine offers, therefore, a possible mechanism for the acyl-remodelling of phosphatidylcholine for the production of lung surfactant.     

Measurement of lysophospholipid acyltransferase activities using substrate competition

Lysophospholipid acyltransferases (LPATs) incorporate fatty acyl chains into phospholipids via a CoA-dependent mechanism and are important in remodeling phospholipids to generate the molecular species of phospholipids found in cells. These enzymes use one lysophospholipid and one acyl-CoA ester as substrates. Traditional enzyme activity assays engage a single substrate pair, whereas in vivo multiple molecular species exist. We describe here an alternative biochemical assay that provides a mixture of substrates presented to the microsomal extracts. Microsomal preparations from RAW 264.7 cells were used to compare traditional LPAT assays with data obtained using a dual substrate choice assay using six different lysophospholipids and eight different acyl-CoA esters. The complex mixture of newly synthesized phospholipid products was analyzed using LC-MS/MS. Both types of assays provided similar results, but the dual choice assay provided information about multiple fatty acyl chain incorporation into various phospholipid classes in a single reaction. Engineered suppression of LPCAT3 activity in RAW 264.7 cells was easily detected by the dual choice method. These findings demonstrate that this assay is both specific and sensitive and that it provides much richer biochemical detail than traditional assays.     

Fatty acid biosynthesis in Erlich cells. The mechanism of short term control by exogenous free fatty acids.

We have examined the mechanism by which extracellular free fatty acids regulate fatty acid biosynthesis in Ehrlich ascites tumor cells. De novo biosynthesis in intact cells was inhibited by stearate greater than oleate greater than palmitate greater than linoleate. The amount of citrate and long chain acyl-CoA in the cells was not changed appreciably by the addition of free fatty acids to the incubation medium, indicating than free fatty acids do not regulate fatty acid biosynthesis by changing the total intracellular content of these metabolites. By measuring the incorporation of labeled free fatty acids into acyl-CoA, however, it was determined that the fatty acid composition of the acyl-CoA poolwas changed dramatically to reflect the composition of the exogenous free fatty acids. The relative inhibitory effects of different free fatty acids appear to depend on the ability of their acyl-CoA derivatives to regulate acyl-CoA carboxylase activity. The acyl-CoA concentration needed to produce 50% inhibition of purified Ehrlich cell carboxylase was found to be 0.68 mum for stearoyl-CoA, 1.6 mum for oleoyl-CoA, 2.2 mum for palmitoyl-CoA, 23 mum for myristoyl-CoA, 30 mum for lauroyl-CoA, and 37 mum for linoleoyl-CoA. In contrast to their effects on de novo synthesis, all of the free fatty acids added except stearate stimulated chain elongation in intact cells. Microsomal chain elongation, the major system for elongation in Ehrlich cells, also was regulated by the composition of the cellular acyl-CoA pool. Lauroyl-CoA, myristoyl-CoA, and palmitoyl-CoA were good substrates for elongation by isolated microsomes; oleoyl-CoA, and linoleoyl-CoA were intermediate; and stearoyl-CoA was a very poor substrate. We conclude that free fatty acids regulate fatty acid biosynthesis by changing the composition of the cellular acyl-CoA pool. These changes control the rate of malonyl-CoA production and, because of the acyl-CoA substrate specificity of the microsomal elongation system, modulate the amount of malonyl-CoA used for chain elongation.     

Acylation of 2-acyl-glycerophosphocholine in guinea-pig heart microsomal fractions.

Acyl-CoA:2-acyl-sn-glycero-3-phosphocholine (GPC) acyltransferase is required for the maintenance of the asymmetric distribution of saturated fatty acids at the C-1 position of phosphatidylcholine; however, this activity has been reported to be absent in cardiac tissue. In the present study a very active acyl-CoA:2-acyl-GPC activity was detected and characterized in guinea-pig heart microsomes (microsomal fractions); the mitochondria did not appear to possess this activity. The acyl-CoA specificity of the microsomal acyl-CoA:2-acyl-GPC acyltransferase was distinct from the corresponding acyl-CoA:1-acyl-GPC acyltransferase. These differences were due to the position of the fatty acid on the lysophospholipid rather than the composition of the fatty acids. The enzyme did not exhibit a distinct preference for saturated fatty acids, as might be expected. Our results suggest that, in the heart, control of the intracellular composition and concentration of acyl-CoAs by acyl-CoA hydrolase and acyl-CoA synthetase may play an important role in maintaining the asymmetric distribution of fatty acids in phosphatidylcholine.     

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p-nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p-nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.     

Enzymatic properties of purified murine fatty acid transport protein 4 and analysis of acyl-CoA synthetase activities in tissues from FATP4 null mice

Fatty acid transport protein 4 (FATP4) is an integral membrane protein expressed in the plasma and internal membranes of the small intestine and adipocyte as well as in the brain, kidney, liver, skin, and heart. FATP4 has been hypothesized to be bifunctional, exhibiting both fatty acid transport and acyl-CoA synthetase activities that work in concert to mediate fatty acid influx across biological membranes. To determine whether FATP4 is an acyl-CoA synthetase, the murine protein was engineered to contain a C-terminal FLAG epitope tag, expressed in COS1 cells via adenovirus-mediated infection and purified to near homogeneity using alpha-FLAG affinity chromatography. Kinetic analysis of the enzyme was carried out for long chain (palmitic acid, C16:0) and very long chain (lignoceric acid, C24:0) fatty acids as well as for ATP and CoA. FATP4 exhibited substrate specificity for C16:0 and C24:0 fatty acids with a V(max)/K(m) (C16:0)/V(max)/K(m) (C24:0) of 1.5. Like purified FATP1, FATP4 was insensitive to inhibition by triacsin C but was sensitive to feedback inhibition by acyl-CoA. Although purified FATP4 exhibited high levels of palmitoyl-CoA and lignoceroyl-CoA synthetase activity, extracts from the skin and intestine of FATP4 null mice exhibited reduced esterification for C24:0, but not C16:0 or C18:1, suggesting that in vivo, defects in very long chain fatty acid uptake may underlie the skin disorder phenotype of null mice.     

Regulation of Triacylglucose Fatty Acid Composition (Uridine Diphosphate Glucose:Fatty Acid Glucosyltransferases with Overlapping Chain-Length Specificity)

UDP-glucose (UDP-Glc):fatty acid glucosyltransferases catalyze the UDP-Glc-dependent activation of fatty acids as 1-O-acyl-[beta]-glucoses. 1-O-Acyl-[beta]-glucoses act as acyl donors in the biosynthesis of 2,3,4-tri-O-acylglucoses secreted by wild tomato (Lycopersicon pennellii) glandular trichomes. The acyl composition of L. pennellii 2,3,4-tri-O-acylglucoses is dominated by branched short-chain acids (4:0 and 5:0; approximately 65%) and straight and branched medium-chain-length fatty acids (10:0 and 12:0; approximately 35%). Two operationally soluble UDP-Glc:fatty acid glucosyltransferases (I and II) were separated and partially purified from L. pennellii (LA1376) leaves by polyethylene glycol precipitation followed by DEAE-Sepharose and Cibacron Blue 3GA-agarose chromatography. Whereas both transferases possessed similar affinity for UDP-Glc, glucosyltransferase I showed higher specificity toward short-chain fatty acids (4:0) and glucosyltransferase II showed higher specificity toward medium-chain fatty acids (8:0 and 12:0). The overlapping specificity of UDP-Glc:fatty acid glucosyltransferases for 4:0 to 12:0 fatty acid chain lengths suggests that the mechanism of 6:0 to 9:0 exclusion from acyl substituents of 2,3,4-tri-O-acylglucoses is unlikely to be controlled at the level of fatty acid activation. UDP-Glc:fatty acid glucosyltransferases are also present in cultivated tomato (Lycopersicon esculentum), and activities toward 4:0, 8:0, and 12:0 fatty acids do not appear to be primarily epidermal when assayed in interspecific periclinal chimeras.     

EFFECT OF FATTY-ACID DOUBLE-BOND POSITION ON THE SELECTIVITY OF RAT-BRAIN ENZYMES: THE INCORPORATION OF OLEIC AND CIS-VACCENIC ACIDS INTO LYSOLECITHIN IN VITRO

Abstract— —Selectivity in the esterification of fatty acids to lysolecithin by rat-brain enzymes in vitro was investigated using free fatty acids (activation plus esterification) and CoA esters (esterification) of two naturally-occurring monoenoic fatty-acid isomers, oleic acid [18:1 (n - 9)] and cis-vaccenic acid [18:1 (n - 7)]. Esterification of free acids to l-acyl-sn-glycero-3-phosphorylcholine (1-acyl GPC) was dependent on CoA and ATP, and was stimulated by MgCl₂ and NaF. Under comparable conditions, fatty-acid activation (acyl-CoA synthetase [acid: CoA ligase (AMP)] EC 6.2.1.3.) appeared to be rate-limiting to 1-acyl GPC acyltransferase (acyl-CoA:l-acylglycero-3-phosphocholine O-acyltrans-ferase, EC 2.3.1.23.), since rates were always less with free fatty acids than with the CoA esters. A comparison of substrate curves obtained with free fatty acids and CoA esters suggests a preference for oleic acid during activation. Acyltransferase activity with 2-acyl GPC was similar with both acyl-CoA isomers, whereas with 1-acyl GPC, activity with oleoyl-CoA consistently exceeded that with cis-vaccenoyl-CoA. This difference between patterns of selectivity in esterification of positions 1 and 2 of lecithin suggests that separate enzymes catalyze the two reactions. The transfer of the isomers to the 2 position was affected in a similar manner by changes in pH and temperature, as well as in protein, fatty acid (or acyl-CoA), and 1-acyl GPC concentrations. Patterns of incorporation with simultaneous incubation of both isomers suggests one enzyme. Differences in acyltransferase activity with the two isomerie acyl-CoA's were observed in subcellular distribution, activity changes with brain maturation, and loss of activity on preincubation of microsomes at 45C. From these results it is not certain whether oleic and cis-vaccenic acids are esterified to the 2 position by separate enzymes, or by one enzyme with different affinities for the isomers. However, the investigation clearly indicates that acyltransferases, and possibly acyl-CoA synthetases in brain possess selectivity related to subtle differences in double-bond position. These selectivities probably are important in determining the specific fatty-acid composition of the complex lipids of brain.     

Purification and some properties of a medium-chain acyl-thioester hydrolase from lactating-rabbit mammary gland which terminates chain elongation in fatty acid synthesis.

1. An acyl-thioester hydrolase was isolated from the cytosol of lactating-rabbit mammary gland. The purified enzyme terminates fatty acid synthesis at medium-chain (C8:0-C12:0) acids when it is incubated with fatty acid synthetase and rate-limiting concentrations of malonyl-CoA. These acids are characteristic products of the lactating gland. 2. The mol.wt. of the enzyme is 29000+/-500 (mean+/-S.D. of three independent preparations), as estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 3. The enzyme also hydrolyses acyl-CoA esters of chain lengths C10:0-C16:0 when these are used as model substrates. The greatest activity was towards dodecanoyl-CoA, and the three preparations had specific activities of 305, 1130 and 2010 nmol of dodecanoyl-CoA hydrolysed/min per mg of protein when 56muM substrate was used. 4. The way in which this enzyme controls the synthesis of medium-chain fatty acids by fatty acid synthetase is briefly discussed.