N-acylation of dog heart ethanolamine phospholipids by transacylase activity

Radioactive N-acylethanolamine phospholipids were produced in dog heart homogenates incubated with acyl-labeled phosphatidylcholine in the presence of 5 mM Ca²⁺ and Triton X-100. 70–80% of the label in the N-acylethanolamine phospholipids was recovered in the N-acyl groups and most of the remainder was in the 1-O-acyl groups. Incubations with 1,2-dipalmitoylPC and 1-palmitoyl-2-linoleoylPC labeled in either the 1-O-acyl or 2-O-acyl moiety showed the predominant utilization of the acyl groups at the sn-1 position, indicating transacylation by phospholipase A₁ (or lysophospholipase) activity. It is suggested that intramolecular transacylation from 1-0-acyl to N-acyl groups of phosphatidylethanolamine also occurred to some extent, thus providing a free primary hydroxy group as an additional acyl acceptor for the transacylation reaction.     

Selectivity of acyl transfer between phospholipids: arachidonoyl transacylase in dog heart membranes

Dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to 1-acyl lysophosphatidylethanolamine. Approximately equal amounts of free fatty acids are produced as well. The reaction exhibits a pH optimum of 7.5-8.5 and does not require Ca2+ or other divalent cations. The reaction proceeds in the absence of exogenous coenzyme A but acyl transfer is enhanced by its addition. The transacylase exhibits a strong preference for arachidonate over linoleate and thus may be involved in the maintenance of the high amounts of arachidonate found in microsomal ethanolamine phospholipids.     

Acylation of monolysocardiolipin in rat heart.

Cardiolipin is a major mitochondrial membrane glycerophospholipid in the mammalian heart. In this study, the ability of the isolated intact rat heart to remodel cardiolipin and the mitochondrial enzyme activities that reacylate monolysocardiolipin to cardiolipin in vitro were characterized. Adult rat heart cardiolipin was found to contain primarily linoleic and oleic acids. Perfusion of the isolated intact rat heart in the Langendorff mode with various radioactive fatty acids, followed by analysis of radioactivity incorporated into cardiolipin and its immediate precursor phosphatidylglycerol, indicated that unsaturated fatty acids entered into cardiolipin mainly by deacylation followed by reacylation. The in vitro mitochondrial acylation of monolysocardiolipin to cardiolipin was coenzyme A-dependent with a pH optimum in the alkaline range. Significant activity was also present at physiological pH. With oleoyl-coenzyme A as substrate, the apparent K(m) for oleoyl-coenzyme A and monolysocardiolipin were 12.5 microm and 138.9 microm, respectively. With linoleoyl-coenzyme A as substrate, the apparent K(m) for linoleoyl-coenzyme A and monolysocardiolipin were 6.7 microm and 59.9 microm, respectively. Pre-incubation at 50 degrees C resulted in different profiles of enzyme inactivation for the two activities. Both activities were affected similarly by phospholipids, triacsin C, and various lipid binding proteins but were affected differently by various detergents and myristoyl-coenzyme A. [(3)H]cardiolipin was not formed from monolyso[(3)H]cardiolipin in the absence of acyl-coenzyme A. Monolysocardiolipin acyltransferase activities were observed in mitochondria prepared from various other rat tissues. In summary, the data suggest that the isolated intact rat heart has the ability to rapidly remodel cardiolipin and that rat heart mitochondria contain coenzyme A-dependent acyltransferase(s) for the acylation of monolysocardiolipin to cardiolipin. A simple and reproducible in vitro assay for the determination of acyl-coenzyme A- dependent monolysocardiolipin acyltransferase activity in mammalian tissues with exogenous monolysocardiolipin substrate is also presented.     

sn-Glycerol-3-phosphate transacylase activity in Euglena gracilis organelles

sn-Glycerol-3-phosphate transacylase activity was demonstrated in Euglena mitochondria, chloroplasts, and microsomes. There was no activity in the 100,000g 1-h supernatant. Exposure of each of the isolated organelles to 1 × 10^?4% Triton X-100 resulted in release of substantial quantities of transacylase activity into the 100,000g supernatant. Products formed by catalysis by the membrane-bound transacylases were heterogenous, while those resulting from catalysis by the extracted enzymes were practically all lysophosphatidate.     

Protection of the ischaemic dog heart by oxfenicine

Myocardial ischaemia was produced in closed-chest anaesthetised dogs by coronary embolisation with Sephadex microspheres. Myocardial enzyme release and lactate production were used to quantitate the severity of ischaemia. In dogs pretreated with oxfenicine (S-4-hydroxyphenylglycine; 0.1mmol.kg^?1) 1h prior to embolisation, peak lactate production (extraction ratio ?25±13%) was significantly less than in control dogs (?72±5%; p < 0.01), and the time post-embolisation at which nett lactate uptake became re-established was reduced from 1.4h to 0.4h. Myocardial release of lactate dehydrogenase isoenzyme 1 (HBDH) up to 5h post-embolisation was reduced by 40% in oxfenicine pretreated dogs and there was a similar reduction in the HBDH arterio-coronary sinus difference. Since oxfenicine pretreatment did not modify the major haemodynamic determinants of myocardial oxygen consumption, we conclude that these beneficial effects result from the ability of oxfenicine to divert myocardial metabolism from fatty acid to carbohydrate oxidation, with a consequent reduction in oxygen demand.     

Acylation of L-asparaginase with total retention of enzymatic activity

Acylation of L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) with complete retention of catalytic activity was achieved. Several parameters of the acylation method, based on the binding of palmitoyl residues to epsilon-NH2 groups of protein, were optimized. The correlation between the acylation degree of L-asparaginase and the retention of catalytic activity was established. For a palmitoyl chloride/protein molar ratio ranging from 50 to 900, a degree of modification of 10 to 30% and a retention of catalytic activity of 98 to 60% respectively, was observed. Hydrophobicity of 30% acylated protein was correlated with turbidity in water and octanol and was compared with the native protein. Acylated protein incorporated into liposomes, showed an increase in catalytic activity in intact form as compared to the native enzyme. By the introduction of a sequential acylation cycle, an improvement of the degree of modification with a maximal value at 50% was obtained. Total retention of catalytic activity was achieved by acylation in the presence of 8 mM L-asparagine in a reactional medium.     

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.     

Phosphodiesterase 3 activity is reduced in dog lung following pacing-induced heart failure

We hypothesized that decreases in expression and/or activity of cAMP-specific phosphodiesterases (PDE) contribute to protective adaptations observed in lung after heart failure. In this study, we compared PDE activity in lung parenchyma isolated from control dogs and those paced to heart failure by assaying cyclic nucleotide hydrolysis in fractions of homogenate supernatant eluted from DEAE-Trisacryl columns. Cyclic nucleotide hydrolysis due to PDE3, PDE4, and PDE5 isoforms was predominant in both control and paced groups. The ratio of PDE3 activity to total cAMP PDE activity was decreased in the paced group compared with control (P < 0.05), whereas PDE4 or PDE5 activity ratios were not different between the two groups. With the use of RT-PCR, message expression for PDE3A or PDE3B did not differ between the two groups. Cilostamide, a selective PDE3 inhibitor, and forskolin, a nonspecific agonist for adenylyl cyclase, both inhibited thapsigargin-induced increases in endothelial permeability in control lung. We conclude that PDE3 activity, but not mRNA expression, is reduced in lung from dogs paced to heart failure, a change that could contribute to heart failure-induced attenuation of the lung endothelial permeability response to injury.     

Acylation of 1-alkenylglycerophosphoethanolamine and 1-acylglycerophosphoethanolamine in guinea-pig heart microsomes

Although the acylation of 1-alkenylglycerophosphocholine in mammalian heart is well documented, the acylation of 1-alkenylglycerophosphoethanolamine in the heart was not reported. In this study, the presence of acyl CoA: 1-alkenylglycerophosphoethanolamine acyltransferase in the guinea pig heart microsomes was demonstrated. 1-Alkenylglycerophosphoethanolamine acyltransferase displayed a high degree of specificity towards acyl-CoA. The order of reactivity with acyl-CoA was found to be: linoleoyl much greater than arachidonyl greater than palmitoyl greater than stearoyl = oleoyl. 1-Acylglycerophosphoethanolamine acyltransferase in the microsomes also exhibited specificity towards acyl-CoA in the following manner: linoleoyl greater than arachidonyl much greater than palmitoyl greater than oleoyl greater than stearoyl. However, such specificity appeared to be dependent on acyl-CoA concentration. The acyl-CoA specificities of both enzymes did not correlate with the C-2 acyl distribution observed in the corresponding microsomal phospholipids. Our results suggest that in addition to the acyl specificity of the acyltransferases, intracellular concentrations of acyl-CoAs may also have an important role in determining the observed acyl patterns of the phospholipids. Based on the acyl specificities, pH profiles, and their responses to heat inactivation and thiol reagents, we conclude that 1-alkenylglycerophosphoethanolamine acyltransferase and 1-acylglycerophosphoethanolamine acyltransferase in guinea-pig heart microsomes are not the same enzyme.     

Effect of lysophosphatidylserine on immunological histamine release

The effect of lysophosphatidylserine on immunological histamine release has been studied in rat peritoneal mast cells actively sensitized with horse serum and in human basophils challenged with anti-IgE. In contrast to other lysophospholipids, lysophosphatidylserine enhances the immunological histamine release in rat mast cells. The effect shows the kinetics of a saturable process with an apparent Km for lysophosphatidylserine of 0.26 microM. A similar Km value (0.21 microM) is found when measuring the non-immunological histamine release activated by lysophosphatidylserine plus nerve growth factor. A comparison with phosphatidylserine shows that a half-maximal response to lysophosphatidylserine occurs at a concentration 4-times lower. In addition, the magnitude of the response is higher. At variance with rat mast cells, lysophosphatidylserine does not influence the histamine release elicited by immunological and non-immunological stimuli in human basophils. The histamine secretion in these cells is instead affected by a calcium ionophore or tetradecanoylphorbolacetate, a compound producing activation of protein kinase C.     

Acylation of lysoglycerophospholipids by adrenal membranes

• 1.1. Substantial differences were found in the acyi donor and lyso-acceptor specificities among subcellular membranes and with respect to different regions of the adrenal gland.
• 2.2. In the presence of Mg²⁺-ATP and CoASH, adrenal microsomes were actively transferring arachidonate to lysophospholipids with acyl acceptor specificity in the order: 1-acyl-GPI > 1-acyl-GPC > 1-acyl-GP. However, when oleoyl-CoA was used, acyi acceptor specificity for the microsomal transferases was in the order: 1-acyl-GPC > 1-acyl-GP > 1-acyl-GPI.
• 3.3. Mitochondrial membranes had very low acyi transfer activity and they preferred 1-acyl-GPC over other lyso-acceptors.
• 4.4. The chromamn granules were apparently lacking this type of activity.

     

A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.     

Emerging roles for lysophosphatidylserine in resolution of inflammation

Despite overlapping structural aspects with other phospholipids, lysophosphatidylserine (lysoPS), the monoacyl derivative of phosphatidylserine (diacylPS), appears to exert unique signaling characteristics important in both the early stages of initiating acute inflammation and in the orchestration of its resolution. LysoPS has long been known as a signaling phospholipid in mast cell biology, markedly enhancing stimulated histamine release and eicosanoid production. More recently, there has been a resurgence of interest in lysoPS as new roles in the promotion of phagocytosis of apoptotic cells, so-called efferocytosis, and resolution of inflammation have been identified. With regard to the latter, lysoPS generated in/on activated or aged apoptotic neutrophils enhances their clearance by macrophages via signaling through the macrophage G-protein coupled receptor G2A. In macrophages, this early acting pathway results in PKA-dependent augmentation of Rac1 activity via increased production of PGE? and cAMP. As such, macrophages stimulated with lysoPS demonstrate significantly increased efferocytic capacity necessary to clear large numbers of recruited neutrophils typical of acute inflammation. Given that clearance of these cells is critical for restoration of tissue function, lysoPS, as a pro-resolving lipid mediator, is hypothesized to play a key role in promoting timely resolution of inflammation. This article will review our current knowledge of lysoPS biology including receptor signaling and mechanisms of generation as well as summarize the more recent evidence of its expanding roles in inflammation.     

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