The acylation of lysophosphoradylglycerocholines in guinea-pig heart mitochondria.
ABSTRACT: The importance of the deacylation-reacylation pathway for attaining the desired fatty acid composition in microsomal phospholipids has been well established. It is not clear, however, whether this mechanism is of equal importance in mitochondria. The absence of acyltransferase activity in mammalian heart mitochondria has been reported in a number of studies. In the present study we report the presence of acyltransferase activities for lysophosphoradylglycerocholines in guinea-pig heart mitochondria. This enzyme showed properties that were considerably different from those of the microsomal enzymes. Of all the acyl-CoAs tested (C18:0, C18:1, C18:2 and C20:4) the mitochondrial enzyme utilized only linoleoyl-CoA as fatty acyl donor and utilized both 1-acyl-sn-glycero-3-phosphocholine and 1-alkenyl-sn-glycero-3-phosphocholine as fatty acyl acceptors. The presence of significant quantities of fatty acids other than linoleate at the C-2 position of mitochondrial acylglycerophosphocholines, coupled with the specificity of the enzyme for linoleoyl-CoA, suggest that, in addition to reacylation, other mechanisms play a significant role in producing the molecular composition of these phospholipids found in the mitochondria.
Project description: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.
Project description:Acyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acyltransferase of human platelets is membrane-bound, has a pH optimum of 7.5, is insensitive to 1 mM-Mg2+, is inhibited by 1 mM-Ca2+, and is stimulated slightly by 1 mM-EDTA. Maximal formation of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine is observed at 150 microM-1-alkyl-sn-glycero-3-phosphocholine and 20 microM unsaturated fatty acyl-CoA. The transfer of unsaturated fatty acyl groups to 1-alkyl-sn-glycero-3-phosphocholine is 3-14 times slower than to 1-acyl-sn-glycero-3-phosphocholine. The CoA esters of linoleate and arachidonate, two unsaturated fatty acyl groups commonly found in platelet phospholipids, are the preferred fatty acyl group donors.
Project description:The developing seeds of Borago officinalis (common borage) accumulate a triacylglycerol oil that is relatively rich in the uncommon fatty acid gamma-linolenate (octadec-6,9,12-trienoic acid). Incubation of developing, whole, cotyledons with [14C]oleate and [14C]linoleate showed that the gamma-linolenate was synthesized by the sequential desaturation of oleate----linoleate----gamma-linolenate. Microsomal membrane preparations from the developing cotyledons contained an active delta 6-desaturase enzyme that catalysed the conversion of linoleate into gamma-linolenate. Experiments were designed to manipulate the [14C]linoleate content of the microsomal phosphatidylcholine. The [14C]linoleoyl phosphatidylcholine labelled in situ was converted into gamma-linolenoyl phosphatidylcholine in the presence of NADH. The substrate for the delta 6-desaturase in borage was, therefore, the linoleate in the complex microsomal lipid phosphatidylcholine, rather than, as in animals, the acyl-CoA. This was further confirmed in experiments that compared the specific radioactivity of the gamma-linolenate, in acyl-CoA and phosphatidylcholine, that was synthesized when [14C]linoleoyl-CoA was incubated with microsomal membranes, NADH and non-radioactive gamma-linolenoyl-CoA. The delta 6-desaturase was positionally specific and only utilized the linoleate in position 2 of sn-phosphatidylcholine. Analysis of the positional distribution of fatty acids in the endogenous microsomal sn-phosphatidylcholine showed that, whereas position 1 contained substantial linoleate, only small amounts of gamma-linolenate were present. The results shed further light on the synthesis of C18 polyunsaturated fatty acids in plants and in particular its relationship to the regulation of the acyl quality of the triacylglycerols in oilseeds.
Project description: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.
Project description:The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.
Project description:Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).
Project description:Lysophospholipases A1 which catalyse the hydrolysis of acyl groups from 1-acylglycerophosphocholine (GPC) have been characterized in a number of mammalian tissues and do not exhibit any acyl specificity. In the present study lysophospholipase activity in guinea-pig heart microsomes (microsomal fractions) that hydrolyses 2-acyl-GPC was detected and characterized. The enzyme showed a high degree of acyl specificity. The relative rates of hydrolysis of individual 2-acyl-GPCs with different fatty acids was as follows: C18:2/C20:1/C18:1/C16:0, 14:6:1:1. When substrates were presented in pairs, the hydrolysis of each substrate by the enzyme was inhibited, but to very different extents. Of each pair of lysolipids examined (2-arachidonoyl- and 2-palmitoyl-GPC; 2-arachidonoyl- and 2-linoleoyl-GPC), the one with the expected higher rate of hydrolysis was more severely inhibited and the degree of inhibition was dependent on the concentration of the other lysolipid. The characteristics of the lysophospholipase A2 suggest the enzyme could work in concert with phospholipase A1 to release arachidonic and linoeic acids for further metabolism. The properties of lysophospholipase A2 and A1 suggest that they are different enzymes.
Project description:In the microsomal fraction from young pea (Pisum sativum L.) leaves, the oleoyl moieties from oleoyl-CoA are principally transferred to the sn-2 position of phosphatidylcholine by oleoyl-CoA:1-acyl-lysophosphatidylcholine acyltransferase. The major product of this acyl transfer is 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine. The 1-palmitoyl(stearoyl)-2-oleoyl phosphatidylcholine is subsequently converted into 1-palmitoyl(stearoyl)-2-linoleoyl phosphatidylcholine by the oleate desaturase complex without equilibrating with the bulk membrane phosphatidylcholine pool. Hence, both the acyl transfer to phosphatidylcholine and the subsequent desaturation of oleoyl moieties occur on the sn-2 position of phosphatidylcholine, and there is also a functional coupling of the acyltransferase and oleate desaturase.
Project description:The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.
Project description: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.