Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons. (1/118)

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-([(4-azidosalicyl)-12-amino)]dodecanoyl-sn-glycerol-3-phospho cholin e (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.  (+info)

Effects of inhibitors of arachidonic acid turnover on the production of prostaglandins by the guinea-pig uterus. (2/118)

The supply of free arachidonic acid from phospholipids is generally regarded as the rate-limiting step for prostaglandin (PG) synthesis by tissues. Two enzymes involved in arachidonic acid uptake into, and release from, phospholipids are acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase A2 (PLA2), respectively. PGF2 alpha produced by the endometrium induces luteolysis in several species including guinea-pigs. Thimerosal, an inhibitor of ACLAT, and aristolochic acid, an inhibitor of PLA2, both reduced, in a concentration-dependent manner, the output of PGF2 alpha from guinea-pig endometrium cultured for 24 h on days 7 and 15 of the oestrous cycle. This study showed that the continual production of PGF 2 alpha by guinea-pig endometrium is not only dependent upon the activity of PLA2 for releasing free arachidonic acid for PGF2 alpha synthesis, but also on the incorporation of arachidonic acid into the phospholipid pool by the activity of ACLAT. The inhibitory effects of thimerosal and aristolochic acid on the outputs of PGE2 and 6-keto-PGF1 alpha were less marked, particularly on day 7 when the low output of PGE2 was unaffected and the output of 6-keto-PGF1 alpha was increased at the lower concentrations of thimerosal. This finding indicates that there are different pools of arachidonic acid bound as phospholipid for the syntheses of PGF2 alpha and 6-keto-PGF1 alpha by guinea-pig endometrium.  (+info)

Inhibition of the plastidial phosphatidylcholine synthesis by silver, copper, lead and mercury induced by formation of mercaptides with the lyso-PC acyltransferase. (3/118)

Plastids greatly rely on the import of extraplastidial precursors for the synthesis of their own lipids, and several studies have shown that a lyso-PC acyltransferase located in the envelope may be involved in the import process. Because the presence of heavy metals in soil or in nutrient solutions induces changes in the lipid composition of plastid membranes (and therefore greatly reduces the photosynthetic capability of plants), we analysed the effect of several metal salts on plastidial lyso-PC acyltransferase activity. Among the 12 heavy metals studied, silver, copper, mercury and lead inhibited this activity. Metal bound to the enzyme was not - or only very slightly - released from the protein except when thiol-reducing agents (and not imidazole) were added. The results strongly suggest that the inhibitory effect is due to a formation of mercaptide between metal and cysteine(s). The relationship between the inhibition of the plastidial lyso-PC acyltransferase activity and the in vivo effects of metal salts on the plastid membranes is discussed.  (+info)

Acylation of lysophosphatidylcholine plays a key role in the response of monocytes to lipopolysaccharide. (4/118)

Mononuclear phagocytes play a pivotal role in the progression of septic shock by producing tumor necrosis factor-alpha (TNF-alpha) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram-negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon-gamma (IFN-gamma), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human monocytic cell line MonoMac 6, the production of TNF-alpha and interleukin-6 (IL-6) was approximately five times greater in cells primed with IFN-gamma than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole-1-yl)heptane phosphonate) and YM 50201 (3-hydroxyethyl 5,3'-thiophenyl pyridine) strongly inhibited (up to 90%) TNF-alpha and IL-6 production in response to LPS in both unprimed MonoMac-6 cells and in cells primed with IFN-gamma. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence-based amplification methods showed that SK&F 98625 inhibited TNF-alpha production by decreasing TNF-alpha mRNA levels in MonoMac-6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.  (+info)

Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking. (5/118)

Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.  (+info)

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferase(s) in human platelets. (6/118)

The activities of acyl-CoA:1-acyl-lysophospholipid acyltransferases (EC 2.3.1.23) have been studied in human platelet lysates by using endogenously formed [14C]acyl-CoA from [14C]fatty acid, ATP and CoA in the presence of 1-acyl-lysophosphatidyl-choline (lysoPC), -ethanolamine (lysoPE), -serine (lysoPS) or -inositol (lysoPI). Linoleic acid as fatty acid substrate had the highest affinity to acyl-CoA:1-acyl-lysophospholipid acyltransferase with lysoPC as variable substrate, followed by eicosapentaenoic acid (EPA) and arachidonic acid (AA). The activity at optimal conditions was 7.4, 7.3 and 7.2 nmol/min per 10(9) platelets with lysoPC as substrate, with linoleic acid, AA and EPA respectively. EPA and AA were incorporated into all lyso-forms. Linoleic acid was also incorporated into lysoPE at a high rate, but less into lysoPS and lysoPI. DHA was incorporated into lysoPC and lysoPE, but only slightly into lysoPI and lysoPS. Whereas incorporation of all fatty acids tested was maximal for lysoPC and lysoPI at 200 and 80 microM respectively, maximal incorporation needed over 500 microM for lysoPE and lysoPS. The optimal concentration for [14C]fatty acid substrates was in the range 15-150 microM for all lysophospholipids. Competition experiments with equimolar concentrations of either lysoPC and lysoPI or lysoPE resulted in formation of [14C]PC almost as if lysoPI or lysoPE were not added to the assay medium.  (+info)

Effect of apoprotein B conformation on the activation of lysolecithin acyltransferase and lecithin: cholesterol acyltransferase. Studies with subfractions of low density lipoproteins. (7/118)

In order to determine the role of apoprotein (apo) B conformation in the activation of the lysolecithin acyl-transferase reaction, we studied the activation of purified enzyme by various subfractions of low density lipoprotein (LDL), isolated by density gradient centrifugation. The activation of LAT correlated positively with the density of LDL and negatively with cholesterol/protein and triglyceride (TG)/protein ratios. The enzyme activation was also positively correlated with the number of trinitrobenzenesulfonic acid-reactive lysine amino groups, which increased with increasing density of LDL. The immunoaffinity of the LDL subfractions for B1B6, a monoclonal antibody directed to the receptor-binding region of apoB, increased with increasing density, while the affinity toward C1.4, a monoclonal antibody directed to the amino-terminal region of apoB, was not altered. Enrichment of normal whole LDL with TG resulted in a 45% reduction in enzyme activation, a 27% decrease in the number of trinitrobenzenesulfonic acid-reactive lysine groups, and a marked reduction in the immunoaffinity for B1B6. All these parameters reversed to normal when the TG-enriched LDL was treated with milk lipoprotein lipase, which specifically reduced the TG content of LDL. The LDL subfractions also supported cholesterol esterification by the purified enzyme, in parallel with lysolecithin esterification, indicating that apoB can also serve as an activator of the lecithin-cholesterol acyltransferase reaction. These results strongly suggest that the localized conformational change of apoB which occurs during the TG depletion of the precursor particle is critical for its activation of acyltransferase reactions, in a manner analogous to its interaction with the cellular receptors.  (+info)

Ypr140wp, 'the yeast tafazzin', displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis. (8/118)

When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.  (+info)

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