Specificity of diacylglycerol acyltransferase from bovine mammary gland, liver and adipose tissue towards acyl-CoA esters.

1. Microsomal diacylglycerol acyltransferase from bovine lactating mammary gland, liver and adipose tissue was capable of acylating microsomal-bound 1,2-dipalmitolyglycerol with acyl-CoA of chain length C4--C18. 2. The activity of the liver and adipose enzymes towards butyryl-CoA and hexanoyl-CoA relative to longer-chain acyl-CoA was similar to that of the mammary enzyme. The Km and V values of the three enzymes with butyryl-CoA and hexanoyl-CoA were similar, except for the V values of the adipose enzyme which were higher. 3. Microsomal diacylglycerol acyltransferase from mammary gland and liver of non-ruminants was also capable of utilizing butyryl-CoA. 4. These results indicate that the unique presence of short-chain acids in ruminant milk triacylglycerols is not caused by differences in specificity between the diacylglycerol acyltransferase from ruminant mammary and other tissues.     

Biosynthesis of triacylglycerols containing short-chain fatty acids in lactating cow mammary gland. Activity of diacylglycerol acyltransferase towards short-chain acyl-CoA esters

1. Microsomal 1,2-diacylglycerol acyltransferase from lactating cow mammary gland incorporated equal molar amounts of microsomal-bound 1,2-dipalmitoyl [2-3H]glycerol and [1-14C]-butyrate, [1-14C]hexanoate or [1-14C]palmitate from their CoA esters into triacylglycerol. The enzyme could also utilize exogenous 1,2-diacylglycerols in the presence of ethanol. 2. The pH optimum of the enzyme was 6.1 and 6.4 with butyryl-CoA and hexanoyl-CoA respectively. Values of V were approximately the same (2.7 and 2.4 nmol-min-1-mg-1, respectively), but values of Km were different (34 and 10 muM, respectively) with these two substrates. Mg2+ was not required as cofactor. 3. The presence ofa Mg2+-dependent phosphatidate phosphatase in the microsomal fraction was demonstrated. 4. It is proposed that triacylglycerols containing butyric and hexanoic acid are biosynthesized in cow mammary gland by the glycerolphosphate pathway, in which long-chain 1,2-diacylglycerols derived from phosphatidic acid are acylated at the sn-3 position by short-chain acyl-CoA esters.     

Purification and properties of a thioesterase from lactating rat mammary gland which modifies the product specificity of fatty acid synthetase

An acyl coenzyme A hydrolase (thioesterase II) has been purified to near homogeneity from lactating rat mammary gland. The enzyme is a monomer of molecular weight 33,000 and contains a single active site residue. The enzyme is specific for acyl groups, as acyl-CoA thioesters, containing eight or more carbon atoms and can also hydrolyze oxygen esters. Thioesterase II is capable of shifting the product specificity of rat mammary gland fatty acid synthetase from predominately long chain fatty acids (C14, C16, and C18) to mainly medium chain fatty acids (C8, C10, and C12). Thioesterase II can restore the capacity for fatty acid synthesis to fatty acid synthetase in which the thioesterase component (thioesterase I) has been inactivated with phenylmethanesulfonyl fluoride or removed by trypsinization. No evidence was found of significant levels of thioesterase II in lactating rat liver. The presence of thioesterase II in the lactating mammary gland and the ability of the enzyme to hydrolyze acyl-fatty acid synthetase thioesters of intermediate chain length, are indicative of a major role for this enzyme in the synthesis of the medium chain fatty acids characteristic of milk fat.     

Biosynthesis of Triglyceride and Other Fatty Acyl Esters by Developing Rat Brain

Abstract: The biosynthesis of triglyceride from 1,2-diglyceride and long-chain acyl coenzyme A (CoA) was studied in developing rat brain. Diglyceride acyltransferase activity was highest in a microsomal fraction, had a neutral pH optimum, and was stimulated by MgCl₂. Palmitoyl CoA and oleoyl CoA served equally well as acyl donors. The enzyme catalyzed the acylation of both endogenous diglyceride and several naturally occurring and synthetic exogenous diglycerides. In addition, short-chain primary and secondary alcohols were found to be acylated under these conditions. A second acylation system, active at low pH, was found to catalyze esterification of ethanol and cholesterol, but not diglyceride, with free fatty acid. These results demonstrate that brain has the capacity to acylate a wide variety of physiological and nonphysiological hydroxyl compounds.     

The synthesis of medium-chain fatty acids by lactating-rabbit mammary gland studied in vitro (Short Communication)

The proportion of C(8:0) and C(10:0) fatty acids synthesized by the microsomal plus particle-free supernatant fraction from lactating rabbit mammary gland is enhanced at high protein concentrations. This fraction appears to contain a soluble high-molecular-weight factor that modifies the specificity of the fatty acid synthetase complex for termination of the growing acyl chain.     

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.     

Stearyl-CoA desaturase of bovine mammary microsomes

Stearyl-CoA desaturase from the microsomal fraction of lactating bovine mammary tissue had a specific activity of 0.4 nmoles oleate formed min^?1 mg^?1 protein. NADH was required for desaturase activity. However, oxidized NAD⁺ and NADP⁺ supported measurable desaturase activity. K_m values for stearyl-CoA and NADH were 25.0 μm and 3.0 μm, respectively. Desaturase was depressed by increasing concentrations of other acyl-CoA esters, i.e., palmityl-CoA and oleyl-CoA (>10 μm). Sn-1,2 diglycerides (1–2.0 μm) depressed desaturase slightly in the order 0–20%, as did l-α-glycerolphosphate (0.2–3.6 μm). 1-Acyl-sn-glycerol-3-phosphorylcholine (>0.1 μm) depressed desaturase activity markedly. Sonication of the microsomal preparation stimulated desaturase activity. The addition of ethanol depressed desaturation, and EDTA inhibited desaturation. Palmityl CoA was equally desaturated by the microsomes. The acyl-CoA desaturase was very stable when stored at ?30 °C as a freeze-dried microsomal preparation, i.e., activity was retained after 12-month storage.Labeled stearate and oleate were isolated as esters (triglycerides and phospholipids) and as free fatty acids, indicating the presence of acyl transferases and acyl-CoA hydrolase in mammary microsomes.     

Substrate selectivity of acyl-CoA:lysolecithin acyltransferase from rabbit lung

The influence of both polar head and acyl chain of lysophospholipid on the activity of partially purified acyl-CoA:lysolecithin acyltransferase from rabbit lung was studied. It was concluded that the presence of methyl groups on the nitrogen of the base was essential for recognition of lysophospholipid as substrate by the enzyme. With respect to the acyl chain length and saturation, the activity followed the order: 16:0 approximately equal to 18:1 greater than 14:0 greater than greater than greater than 18:0 approximately equal to 12:0. Also, the effect on the activity of the acyl chain on acyl-CoA was studied. The activity showed great selectivity for saturated acyl-CoAs. The activity with polyunsaturated fatty acids was very low and in the case of arachidonoyl-CoA was almost negligible. The comparison between crude microsomal preparations and partially purified preparations allowed to suggest that it could exist two different acyl-CoA:lysolecithin acyltransferases differing in their selectivity towards saturated and unsaturated fatty acids.     

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.     

Biosynthesis of Glucosyl Diglycerides by Mycoplasma laidlawii Strain B

Monoglucosyl diglyceride is synthesized from 1,2-diglyceride and uridine-5'-diphosphoglucose (UDP); diglucosyl diglyceride from monoglucosyl diglyceride, and uridine-5'-diphosphoglucose by membranes of Mycoplasma laidlawii strain B. All of these enzymatic activities reside in the membrane. Membranes solubilized by detergent action or succinylation and acetone powders of membranes were inactive. Requirements for Mg(2+), UDP, and appropriate lipid acceptor were demonstrated for biosynthesis of both glycolipids. Glucose-1-phosphate plus uridine triphosphate could replace the UDP requirement. A medium of relatively high ionic strength and a critical concentration of sodium lauryl sulfate stimulated biosynthesis of the monoglucosyl diglyceride. The optimal pH for both reactions was 8.0. A specificity for 1,2-diglyceride from the homologous organism was found for optimal synthesis of the monoglucosyl diglyceride, and a specificity for monoglucosyl diglyceride was found in the case of diglucosyl diglyceride synthesis. Both reactions were specific for UDP.     

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.     

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins1

Liver and intestinal cytosol contain abundant levels of long chain fatty acyl-CoA binding proteins such as liver fatty acid binding protein (L-FABP) and acyl-CoA binding protein (ACBP). However, the relative function and specificity of these proteins in microsomal utilization of long chain fatty acyl-CoAs (LCFA-CoAs) for sequential transacylation of glycerol-3-phosphate to form phosphatidic acid is not known. The results showed for the first time that L-FABP and ACBP both stimulated microsomal incorporation of the monounsaturated oleoyl-CoA and polyunsaturated arachidonoyl-CoA 8–10-fold and 2–3-fold, respectively. In contrast, these proteins inhibited microsomal utilization of the saturated palmitoyl-CoA by 69% and 62%, respectively. These similar effects of L-FABP and ACBP on microsomal phosphatidic acid biosynthesis were mediated primarily through the activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting step, rather than by protecting the long chain acyl-CoAs from microsomal hydrolase activity. In fact, ACBP but not L-FABP protected long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity in the order: palmitoyl-CoA>oleoyl-CoA>arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.     

The pattern of fatty acid synthesis in lactating rabbit mammary gland studied in vivo

The biosynthesis of fatty acids has been studied in lactating rabbits at 6h after intravenous injection of sodium [1-(14)C]acetate. The specific radioactivities of the individual fatty acids (C(6:0) to C(14:0)) and the proportions of these fatty acids synthesized were similar in mammary tissue and milk. Hexanoic acid had the highest specific radioactivity, and the C(8:0)-C(14:0) fatty acids had similar specific radioactivities, which were about five times those of C(16) and C(18) acids. No radioactivity was detected in fatty acids of chain length C(14) in these tissues were similar to those of the long-chain fatty acids in the milk and mammary gland. The results show that the C(4:0)-C(14:0) fatty acids are synthesized within the mammary gland rather than by fatty acid uptake from circulating blood or by oxidation of long-chain fatty acids within the gland. We conclude that de novo synthesis of esterified fatty acids in vivo by this tissue has a high degree of chain-length specificity.     

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.     

The structure of a glycerylphosphoryldiglucosyl diglyceride from the lipids of Acholeplasma laidlawii strain B

1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6'-[O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha- d-glucopyranosyl-(1-->1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.     

Modification of seed oil content and acyl composition in the brassicaceae by expression of a yeast sn-2 acyltransferase gene.

A putative yeast sn-2 acyltransferase gene (SLC1-1), reportedly a variant acyltransferase that suppresses a genetic defect in sphingolipid long-chain base biosynthesis, has been expressed in a yeast SLC deletion strain. The SLC1-1 gene product was shown in vitro to encode an sn-2 acyltransferase capable of acylating sn-1 oleoyl-lysophosphatidic acid, using a range of acyl-CoA thioesters, including 18:1-, 22:1-, and 24:0-CoAs. The SLC1-1 gene was introduced into Arabidopsis and a high erucic acid-containing Brassica napus cv Hero under the control of a constitutive (tandem cauliflower mosaic virus 35S) promoter. The resulting transgenic plants showed substantial increases of 8 to 48% in seed oil content (expressed on the basis of seed dry weight) and increases in both overall proportions and amounts of very-long-chain fatty acids in seed triacylglycerols (TAGs). Furthermore, the proportion of very-long-chain fatty acids found at the sn-2 position of TAGs was increased, and homogenates prepared from developing seeds of transformed plants exhibited elevated lysophosphatidic acid acyltransferase (EC 2.3.1.51) activity. Thus, the yeast sn-2 acyltransferase has been shown to encode a protein that can exhibit lysophosphatidic acid acyltransferase activity and that can be used to change total fatty acid content and composition as well as to alter the stereospecific acyl distribution of fatty acids in seed TAGs.     

Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity

The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase.     

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.     

Coenzyme A-dependent transacylation system in rabbit liver microsomes

The activities of cofactor-independent and CoA-dependent transacylation were examined for various rabbit tissues. Liver microsomes were found to exhibit relatively high CoA-dependent transacylation activity, while the cofactor-independent transacylation activity was low. The apparent Km values for CoA were 1.4 microM (acceptor, 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC] and 3.8 microM (acceptor, 1-acyl-sn-glycero-3-phosphoethanolamine (1-acyl-GPE], respectively. The apparent Vmax values were 2.6 nmol/min/mg (1-acyl-GPC) and 1.2 nmol/min/mg (1-acyl-GPE), respectively. The CoA-dependent transacylation reaction shows a distinct fatty acid specificity. [14C]18:2 and [14C]20:4 at the 2-positions and [14C]18:0 at the 1-positions of donor phospholipids were transferred to lysophospholipids in the presence of CoA. We observed the formation of considerable amounts of acyl-CoA from these fatty acids during the reaction, without the participation of ATP. The transfer of other fatty acids between phospholipids was shown to be almost nil. The very low transfer of 18:1 was in marked contrast to the effective utilization of 18:1-CoA by acyl-CoA:1-acyl-GPC acyltransferase. The effects of several compounds and heat treatment on these two acylation reactions were also examined. The CoA-dependent transacylation reaction may be important for the selective acylation of certain lysophospholipids, such as 1-acyl-GPE, in living cells with the cooperation of acyl-CoA:lysophospholipid acyltransferase, which generates CoA for the former reaction.     

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.