Acylation of lysophospholipids by rabbit alveolar macrophages. Specificities of CoA-dependent and CoA-independent reactions

Intact alveolar macrophages were found to acylate alkyl- and acyllysophospholipids with a high selectivity for arachidonate. A specific mechanism appears responsible for the incorporation of arachidonate into lysophospholipids in intact cells since the kinetic pattern for the formation of the 20:4 species was different from all other species. This specificity was investigated in more detail by examining the enzymatic acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by macrophage membranes; in the absence of CoA, ATP, and Mg2+, this lysophospholipid was acylated with a high preference for arachidonate that was independent of added free fatty acids. The addition of CoA alone increased the rate of acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine, mainly due to an increase in the formation of species other than those containing arachidonate. When CoA, ATP, and Mg2+ were present, the macrophage membranes catalyzed the acylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine without preference for arachidonate. A different apparent Km and Vmax was observed for reactions involving each cofactor condition. We conclude that the acylation of alkyl- and acyllysophospholipids by rabbit alveolar macrophages occurs by three separate mechanisms: a CoA-independent transacylation, a CoA-dependent transacylation (reverse reaction catalyzed by acyl-CoA acyltransferase), and an acyl-CoA-dependent acylation. The CoA-independent transacylation reaction is unique in that it is specific for arachidonate and accounts for the selective acylation of alkyl- and acyllysophospholipids by arachidonate in membrane preparations of alveolar macrophages. This reaction appears to be extremely important in the remodeling of phospholipid molecular species and the mobilization of arachidonate into ether-linked lipids. The transfer of arachidonate to 1-alkyl-2-lyso-sn-glycero-3-phosphocholine also is of importance in the final inactivation step for platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine), whereby 1-alkyl-2-arachidonoyl-sn-glycerol-3-phosphocholine (a stored precursor of both platelet activating factor and arachidonic acid metabolites) is formed.     

Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms

Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme’s genes are described.     

Transacylation of lyso platelet-activating factor and other lysophospholipids by macrophage microsomes. Distinct donor and acceptor selectivities

Rabbit alveolar macrophage microsomes were found to acylate 1-[3H]alkyl-glycero-3-phosphocholine (GPC) (lyso platelet-activating factor) in the absence of any cofactors, indicating the presence of transacylation activity. The transacylation activity was comparable to the activity of acyl-CoA:1-alkyl-GPC acyltransferase. The fatty acyl moieties introduced into 1-[3H]alkyl-GPC from membrane lipids by microsomes were mainly 20:4 (n-6). A very similar acylation profile was observed for the acylation of 1-[3H]alkyl-GPC in intact macrophages, suggesting that the CoA-independent transacylation system plays a very important part in the acylation of 1-[3H]alkyl-GPC in cells. We also confirmed that 14C-labeled 20:4(n-6), 20:5(n-3), 22:4(n-6), and 22:6(n-3) were transferred well from diacyl-GPC to 1-alkyl-GPC in a CoA-independent manner. The transfer rates for 16:0, 18:0, and 18:1 from diacyl-GPC to 1-alkyl-GPC were very low in the presence and absence of CoA. On the other hand, the transfer of 20:4 from diacyl-GPE or diacyl-GPI to 1-alkyl-GPC or 1-acyl-GPC was markedly increased by the addition of CoA. The above results indicate that the transacylation system exhibits distinct donor and acceptor selectivities and CoA dependency. These transacylation reactions could be very important in the regulation of the levels and the availability of lysophospholipids, including lyso platelet-activating factor, and C20 and C22 polyunsaturated fatty acids in living cells.     

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.     

Very high proportion of disaturated molecular species in rat platelet diacyl-glycerophosphocholine: involvement of CoA-dependent transacylation reactions

The molecular species composition of rat platelet diacyl-glycerophosphocholine (GPC) was investigated by reverse-phase HPLC and by mass spectrometry. The two methods gave the same very high proportion of fully saturated phospholipids, the 16:0-16:0 and 16:0-18:0 species representing together about 40% of the overall molecular species. [14C]Palmitoyllyso-GPC was found to be acylated by resting platelets in equal amounts into 16:0-16:0 and into 16:0-20:4 species. The acylation rate of this lysophospholipid was increased by 3-fold and 14-fold when platelets were stimulated for 10 min with thrombin and the ionophore A23187, respectively. Essentially the same two molecular species were synthesized upon stimulation but with a higher preference for arachidonate than for palmitate. We investigated the mechanisms responsible for the incorporation of palmitate and arachidonate by examining the enzymatic acylation of [14C]palmitoyllyso-GPC by platelet homogenates. The percentage of the various molecular species formed when CoA, ATP, and Mg2+ were added excludes the CoA, ATP-dependent pathway as being involved in the acylation reactions previously observed. In the absence of ATP, CoA-independent transacylations appear to play a crucial role in the synthesis of the 16:0-20:4 species whereas the addition of CoA greatly favored dipalmitoyl-GPC synthesis. The involvement of CoA-dependent mechanisms in the synthesis of dipalmitoyl-GPC was demonstrated as follows: (i) the labeling in the sn-2 position of the dipalmitoyl-GPC synthesized in the presence of CoA was not modified when free unlabeled palmitic acid was added to the incubation medium and (ii) platelet homogenates were unable to esterify lysolecithin with added labeled palmitic acid in the presence of CoA only.     

Biosynthesis of platelet activating factor. Substrate specificity of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase in rat spleen microsomes

The substrate requirements and specificity of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC):acetyl-CoA acetyltransferase were investigated. The following findings were observed. 1) When the ether bond of alkyllyso-GPC is substituted with an ester linkage, the resulting compound, palmitoyllyso-GPC, can serve as a substrate, albeit at a reduced rate (50%). In addition, palmitoyllyso-GPC is a competitive inhibitor in the reaction with respect to concentration dependence of alkyllyso-GPC and a noncompetitive inhibitor when the concentrations of acetyl-CoA are varied. 2) Octadecyllyso-GPC is acetylated at a slightly higher rate than hexadecyllyso-GPC and unsaturated alkyllyso-GPC is a preferable substrate to its saturated counterpart. 3) The homologous series of short chain acyl-CoAs demonstrate an inverse relationship of chain length with the values of their apparent Km and Vmax, e.g. the longer the acyl-CoA chain, the smaller the values of Vmax and apparent Km. 4) The effect of polar head group modification of alkyllyso-GPC on the acetyltransferase activity is related to the degree of methylation of the amine group. The choline base analog gives the highest enzyme activity and the ethanolamine derivative is the least active, while N', N'-dimethylethanolamine and monomethylethanolamine analogs are the substrates with intermediate activities. These results on substrate selectivity of acetyltransferase correlate with the known structural requirements essential for the biological activities elicited by platelet activating factor and thus suggest that the acetyltransferase activating factor and thus suggest that the acetyltransferase may be important in governing the chemical structure of platelet activating factor synthesized in vivo.     

Existence of endogenous inhibitors of platelet-activating factor (PAF) with PAF in rat uterus

Two kinds of phospholipids in normal rat uterus were found to inhibit the aggregation of washed rabbit platelets induced by 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC) and were named Inhibitor I and Inhibitor II and identified by mass spectrometry. Inhibitor I was a mixture of 1-acyl (16:0, 18:0, 18:1, 18:2, and 20:4)-2-lyso-sn-glycero-3-phosphocholine (acyllyso-GPC) and 1-alkyl (16:0, 18:0, and 18:1)-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC). 16:0 acyllyso-GPC was the most inhibitory, followed by 18:1, 18:2, 20:4, and 18:0 acyllyso-GPCs and 16:0 alkyllyso-GPC. Their IC50 values were in the range of 1-4 X 10(-5) M against the platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC, indicating that they were about 100 times weaker inhibitors than CV-3988. Inhibitor II was a mixture of N-acyl sphing-4-enyl phosphocholine (18:1/18:0, 18:1/20:0, 18:1/24:0, and 18:1/24:2). The most inhibitory of these components were 18:1/20:0 and 18:1/24:0, followed by 18:1/24:2 and 18:1/18:0, and their IC50 values were in the range of 4-5 X 10(-5) M against platelet aggregation induced by the alkylacetyl-GPC. Quantitatively, about 10(5) times higher concentrations of these inhibitors should be necessary to inhibit platelet aggregation induced by 1 X 10(-10) M 16:0 alkylacetyl-GPC. In fact, the contents of Inhibitors I and II, respectively, were approximately 10(5) times (4.7 X 10(-2) and 7.1 X 10(-2) mol/mol lipid-phosphorus of the original uterine phospholipids) than that of 16:0 alkylacetyl-GPC (1.4 X 10(-6) mol/mol lipid-phosphorus). The role of alkylacetyl-GPC in normal rat uterus is uncertain, but it coexists in situ with two kinds of endogenous inhibitors, choline containing lysoglycerophospholipids and sphingophospholipids.     

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.     

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil

Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [³H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [³H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [³H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTPγS and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [³H]alkylacyl-GP and [³H]alkylacyl-GPethanol, phospholipase D breakdown products from [³H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [³H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes. © 1996 Wiley-Liss, Inc.     

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.     

Metabolism of platelet-activating factor in human platelets. Transacylase-mediated synthesis of 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine

The present study demonstrates that inactivation of exogenous 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC; platelet-activating factor) by human platelets is mediated by the sequential action of two enzymes, 1) a Ca2+-independent acetylhydrolase recovered in the cytosolic fraction of platelets that deacylates alkylacetyl-GPC forming alkyllyso-GPC and 2) a CoA-independent, N-ethylmaleimide-sensitive transacylase associated with platelet membranes that incorporates a long-chain fatty acid into alkyllyso-GPC to produce alkylacyl-GPC. Separation of platelet phospholipids and subsequent resolution into individual molecular species by high-performance liquid chromatography revealed that the newly formed alkylacyl-GPC was exclusively alkylarachidonoyl-GPC and that the arachidonoyl group for acylation of alkyllyso-GPC was provided by phosphatidylcholine. We conclude that the previously described platelet arachidonoyl transacylase (Kramer, R.M., and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811) may play an important role in the metabolism of platelet-activating factor.     

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.     

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

Summary Molecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C_36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C_38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A₂, in addition to other metabolic processes, in mediating these changes is discussed.Abbreviations ACL average chain length - UI unsaturation index     

Phosphatidyl choline fatty acid remodeling in the hepatic cell nuclei

This study was performed to determine whether fatty acids incorporated into liver cell nuclei phosphatidylcholine (PtdCho) could be remodeled in the isolated nuclear. For this reason, rat liver cell nuclei were incubated in vitro with [1-14C]20:4n-6-CoA. PtdCho molecular species with the highest specific activity had an unsaturated fatty acid at sn-1 and sn-2 positions (20:4-20:4>18:2-20:4>18:1-20:4). 16:0-20:4 and 18:0-20:4 PtdChos showed a minor specific activity. When labeled nuclei were reincubated in the absence of labeled substrate with the addition of cytosol, ATP and CoA, the specific activity of 20:4-20:4, 18:2-20:4 and 18:1-20:4 species decreased, while that of 16:0-20:4 and 18:0-20:4 increased. In conclusion, the asymmetric fatty acid distribution of saturated fatty acids at sn-1 position, and unsaturated fatty acids at sn-2 position of nuclear PtdCho molecular species was re-established by an acyl-CoA-dependent remodeling process.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.     

Influence of dietary n-3 fatty acids on macrophage glycerophospholipid molecular species and peptidoleukotriene synthesis

The study examined the ability of dietary n-3 fatty acids to modify mouse peritoneal macrophage glycerophospholipid molecular species and peptidoleukotriene synthesis. After a 2-week feeding period, fish versus corn oil feeding significantly (P less than 0.01) lowered n-6 polyunsaturated fatty acid (PUFA) mol % levels, i.e., arachidonic acid (20:4n-6) in diacylphosphatidylserine (PtdSer), diacylphosphatidylinositol (PtdIns), diacylglycerophosphoethanolamine (PtdEtn), alkenylacylglycerophosphoethanolamine (PlsEtn), and diacylglycerophosphocholine (PtdCho). A notable exception was alkylacylglycerophosphocholine (PakCho), where only moderate decreases in 16:0-20:4n-6 and 18:0-20:4n-6 species were observed after fish oil supplementation. The predominant n-3 PUFA in macrophage phospholipid subclasses was docosapentaenoic acid (22:5n-3). The major n-3 species were 18:0-22:5n-3 in PtdIns, PtdSer, glycerophosphoethanolamines (EtnGpl) and 16:0-22:5n-3 in PtdCho and PlsEtn. The major n-3-containing species in PakCho were 16:0-20:5n-3 and 18:1-22:6n-3. These findings indicate that n-3 PUFA are differentially incorporated into macrophage phospholipid subclasses after dietary fish oil supplementation, and suggest that phospholipid remodeling enzymes selectively discriminate between substrates based on compatibility of sn-1 covalent linkage and the composition of the sn-1 and sn-2 aliphatic chains. Macrophage peptidoleukotriene synthesis was also strongly influenced after fish oil feeding; the LTC5/LTC4 ratio was significantly higher (P less than 0.01) in fish oil-fed animals than in corn oil-fed animals, 0.85 versus 0.01, respectively. These ratios were subsequently compared to phospholipid molecular species 20:5n-3/20:4n-6 ratios in order to determine potential sources of eicosanoid precursors.     

Interrelationships between the enzymes of ethanolamine metabolism in Escherichia coli

The activities of the enzymes ethanolamine ammonia-lyase, CoA-dependent and CoA-independent aldehyde dehydrogenases, and isocitrate lyase were assayed in Escherichia coli which had been grown on various sources of carbon and nitrogen. Induction of ethanolamine ammonia-lyase and of maximal levels of both aldehyde dehydrogenases required the concerted effects of ethanolamine and vitamin (or coenzyme) B12. Molecular exclusion chromatography revealed that, in the absence of one or both co-inducers, two repressible isoenzymes of CoA-dependent aldehyde dehydrogenase (mol. wts 900000 and 120000) were produced, these being replaced by two inducible isoenzymes (mol. wts 520000 and 370000) in the presence of both co-inducers. A similar inducible repressible series of isoenzymes was also observed for CoA-independent aldehyde dehydrogenase. No evidence was found for structural relationships between ethanolamine ammonia-lyase, CoA-dependent aldehyde dehydrogenase and CoA-independent aldehyde dehydrogenase, but mutant and physiological studies demonstrated that the induction of the first two enzymes is under common control. Evidence is presented for the operation of a previously unreported pathway of ethanolamine metabolism in E. coli.     

Molecular species specificity of phospholipid breakdown in microsomal membranes of senescing carnation flowers

During senescence of cut carnation flowers, there is extensive breakdown of microsomal phospholipid. This is attributable, at least in part, to lipolytic activity associated directly with the microsomal membranes. Evidence indicating that one or more of the lipid-degrading enzymes in these membranes preferentially degrade phospholipid molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain has been obtained by using radiolabeled phosphatidylcholine substrates. 16:0^*/16:0^*, 16:0/18:2^*, and 18:1^*/18:1^* phosphatidylcholine were degraded only minimally over a 3 hour period by microsomes isolated from senescing flowers. By contrast, [U-¹⁴C]phosphatidylcholine, which comprises various molecular species including those containing polyunsaturated acyl chains, and 18:0/20:4^* phosphatidylcholine were extensively degraded. Under identical conditions, but in the absence of added radiolabeled substrate, endogenous 18:2/18:2, 18:1/18:3, and 18:2/18:3 phosphatidylcholine were selectively depleted from the membranes. During natural senescence of the flowers, there was a sharp decline in microsomal 16:0/18:1 and 18:1/18:2 phosphatidylcholine, whereas molecular species containing two diunsaturated acyl chains or at least one polyunsaturated acyl chain remained unchanged or decreased only slightly. The data have been interpreted as indicating that provision of particular molecular species susceptible to lipase attack is a prerequisite to phospholipid catabolism in senescing membranes.     

Molecular Species of Diacylglycerols and Phosphoglycerides and the Postmortem Changes in the Molecular Species of Diacylglycerols in Rat Brains

The molecular species of 1,2-diacyl-sn-glycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) from brains of adult rats (weighing 150 g) were determined. The DAG, isolated from brain lipid extracts by TLC, was benzoylated, and the molecular species of the purified benzoylated derivatives were separated from each other by reverse-phase HPLC. The total amount and the concentration of each species were quantified by using 1,2-distearoyl-sn-glycerol (18:0-18:0) as an internal standard. About 30 different molecular species containing different fatty acids at the sn-1 and sn-2 positions of DAG were identified in rat brains (1 min postmortem), and the predominant ones were 18:0-20:4 (35%), 16:0-18:1 (15%), 16:0-16:0 (9%), and 16:0-20:4 (8%). The molecular species of PC, PE, PS, and PI were determined by hydrolyzing the lipids with phospholipase C to DAG, which was then benzoylated and subjected to reverse-phase HPLC. PIP and PIP2 were first dephosphorylated to PI with alkaline phosphatase before hydrolysis by phospholipase C. The molecular species composition of phosphoinositides showed predominantly the 18:0-20:4 species (50% in PI and approximately 65% in PIP and PIP2). PS contained mainly the 18:0-22:6 (42%) and 18:0-18:1 (24%) species. PE was mainly composed of the 18:0-20:4 (22%), 18:0-22:6 (18%), 16:0-18:1 (15%), and 18:0-18:1 (15%) species. In PC the main molecular species were 16:0-18:1 (36%), 16:0-16:0 (19%), and 18:0-18:1 (14%). Studies on postmortem brains (30 s to 30 min) showed a rapid increase in the total amount (from 40-50 nmol/g in 0 min to 210-290 nmol/g in 30 min) and in all the molecular species of DAG. Comparatively larger increases (seven- to 10-fold) were found for the 18:0-20:4 and 16:0-20:4 species. Comparison of DAG species with the molecular species of different glycerolipids indicated that the rapid postmortem increase in content of DAG was mainly due to the breakdown of phosphoinositides. However, a slow but continuous breakdown of PC to DAG was also observed.     

Effect of various temperatures on phosphatidylglycerol biosynthesis in thylakoid membranes

Lipid unsaturation, the major factor to maintain thylakoid membrane fluidity, is affected by temperature. In this work, we analysed the molecular species composition of phosphatidylglycerol (PG) in thylakoid membranes during spinach ( Spinacia oleracea ) and squash ( Cucurbita pepo ) cotyledon growth to investigate how the growth temperature affects the PG biosynthesis. Of the 10 molecular species detected, temperature affected mainly the relative content of molecular species containing linolenic acid (18:3) and those containing palmitic acid (16:0) at the sn -1 position of glycerol backbone. Lowering the temperature induced an increase in the former and a decrease in the latter. The relative content of molecular species containing 18:3 or 16:0 at the sn -1 position of the glycerol backbone were correlated with temperature. Our results indicate that the substrate selectivity of the glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts towards 16:0 or oleic acid (18:1) and the activity of fatty acid desaturases are greatly affected by temperature. In addition, changes in the relative content of PG molecular species induced by variations in growth temperature depended mainly on the substrate selectivity of GPAT.