Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism最新文献

The Group VI 80-kDa Ca²⁺-independent phospholipase A₂ (iPLA₂) has been purified from murine P388D₁ macrophages and Chinese hamster ovary (CHO) cells. The amino acid sequence of the iPLA₂ has been determined and shown to contain a lipase consensus sequence and eight ankyrin repeats, which makes it distinct from Group I–V PLA₂s. This enzyme appears to play a key role in mediating basal phospholipid remodeling. We now report that the Group VI iPLA₂ displays interfacial activation toward short chain phospholipids, 1-octanoyl-2-heptanoyl-sn-glycero-3-phosphocholine, 1,2-diheptanoyl-sn-glycero-3-phosphocholine, and 1,2-dihexanoyl-sn-glycero-3-phosphocholine micelles. ATP protects the iPLA₂ from a loss in activity as a result of prolonged incubation during the assay. Hence higher enzyme activity is observed in the presence than in the absence of ATP. Similar protection was obtained with glycerol. In addition, the iPLA₂ exhibits multiple activities which are strongly dependent on substrate presentation. The lysophospholipase activity of this enzyme was diminished by Triton X-100 and stimulated by glycerol. With the combination of 50 μM Triton X-100 and 50% glycerol, the enzyme’s lysophospholipase activity achieved equivalent activity to its PLA₂ activity. The iPLA₂ displayed both lysophospholipid/transacylase and phospholipid/transacylase activity, supporting the conclusion that the mechanism of action of iPLA₂ proceeds through an acyl-enzyme intermediate as proposed for the Group IV cPLA₂.

The inhibitory effects of MgATP on neuronal nuclear acetyltransferase activities were studied using lyso platelet-activating factor (lyso-PAF, 1-alkyl-sn-glycero-3-phosphocholine) and lysophosphatidylcholine (lyso-PC, 1-acyl-sn-glycero-3-phosphocholine). The nuclear (N₁) acetylation of lyso-PC was more profoundly inhibited by MgATP. MgATP did not alter the apparent K_m for acetyl-CoA in either acetylation reaction. The inhibitory effects of MgATP were not seen for other nucleotides or MgAMP-PCP. Kinase inhibitors such as staurosporine (1 μM), chelerythrine, and R59022 (diglyceride kinase inhibitor I) did not block the MgATP inhibition of either acetylation. However, the addition of phospholipids to the assays indicated a selective inhibitory effect for PIP (25–50 μM) in the nuclear acetylation of lyso-PAF. When N₁ was incubated with [γ-³³P]ATP, phosphatidic acid and PIP were the principal radioactive lipid products. While the extent of MgATP inhibition of lyso-PAF acetylation was similar at different concentrations of lyso-PAF, increasing lyso-PC concentrations greatly decreased the MgATP inhibition seen in lyso-PC acetylations. Nuclear envelopes prepared in the presence of PMSF, and fraction N₁ exposed to PMSF, did not show the inhibitory effect of MgATP on lyso-PC acetylation. PMSF (an inhibitor of certain phospholipase and lysophospholipase activities) did not reduce the MgATP inhibition of lyso-PAF acetylation. Arachidonoyl trifluoromethylketone, an inhibitor of cytosolic phospholipases A₂ and of lysophospholipase activity associated with cPLA₂, also blocked the inhibitory effect of MgATP on lyso-PC acetylation. Using radioactive lyso-PC substrate, fraction N₁ produced labeled free fatty acid and phosphatidylcholine. In the presence of acetyl-CoA, the production of radioactive phosphatidylcholine increased almost 6-fold when MgATP was also included in these incubations. In the presence of MgATP and acetyl-CoA, PMSF reduced the levels of radioactive free fatty acid and phosphatidylcholine derived from lyso-PC, while Triacsin C, an inhibitor of acyl CoA synthetase, decreased phosphatidylcholine labeling. These findings suggest that MgATP inhibition of lyso-PC acetylation results from a loss of lyso-PC substrate that is largely mediated by nuclear lysophospholipase, acyl-CoA synthetase and lyso-PC acylation. Thus the neuronal nuclear production of Acyl PAF may be regulated by paths that compete for the lyso-PC substrate. In contrast, the acetylation of lyso-PAF is inhibited by PIP, a product of nuclear PI kinase reactions.

Rats were fed on a low fat diet or on high fat diets which included coconut oil, olive oil, safflower oil, evening primrose oil or fish oil as the principal fat source. The level of phosphatidylinositol-4,5-bisphosphate in spleen lymphocytes was unaffected by diet. However, the fish oil diet significantly decreased the concentration of inositol-1,4,5-trisphosphate in stimulated lymphocytes; this concentration was also reduced following olive oil feeding. Diet did not significantly affect the level of phospholipase C-γ1 in spleen lymphocytes but the tyrosine phosphorylation state of this enzyme in stimulated lymphocytes, as well as that of a range of other proteins, was decreased following feeding the fish oil and, to a lesser extent, the olive oil diets. It is concluded that fish oil feeding appears to result in inhibition of one or more tyrosine kinases.

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100 000×g membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase (S.t.LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t.LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase (S.t.LOX) by using different polyunsaturated fatty acids as substrates.

Tumor necrosis factor α (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G₀+G₁/S, accompanied by apoptosis. ³¹P and ¹³C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and ²H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52±6%. Later (∼15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis.

The sphingolipid sulfatide is a component of myelin and some non-neuronal cells. Antibodies to sulfatide occur in some patients with autoimmune neuropathies and in patients with insulin-dependent diabetes mellitus (IDDM) caused by immunologic destruction of insulin-secreting pancreatic islet β-cells. Distinct sulfatide molecular species may differ in immunogenicity, and facile means to identify sulfatide species in islets and other tissues obtainable in only small amounts could be useful. Electrospray ionization mass spectrometry (ESI/MS) permits structural determination of small quantities of phospholipids and is applied here to sulfatide analysis. We find that sulfatide standards are readily analyzed by negative ion ESI/MS, and tandem mass spectra of individual species exhibit some ions common to all species and other ions that reflect distinct fatty acid substituents in different sulfatide molecules. A signature ion cluster resulting from cleavage directed by the α-hydroxy group of sulfatide species with a hydroxylated fatty acid substituent identifies such species. Sulfatide profiles in tissue lipid extracts can be obtained by ESI/MS/MS scanning for common sulfatide ions and for ions reflecting fatty acid substituents. Islets are demonstrated to contain sulfatide and to exhibit a profile of species different from that of brain.

The enzyme responsible for most of the phospholipase A₂ (PLA₂) activity present in bovine seminal plasma was recently purified to homogeneity. Sequencing revealed that the enzyme is also a platelet-activating factor acetylhydrolase (PAF-AH) of the serum type with kinetic properties generally similar to its serum homologue. In the present work, we have attempted to clarify its physiological function by studying its association properties in seminal plasma. As was observed previously for its PLA₂ activity, its PAF-AH activity was also inhibited by the major proteins of bovine seminal plasma (BSP proteins). Sequential dilution experiments as well as centrifuging semen on Percoll did not reveal detectable association of PAF-AH with spermatozoa. Neither did the enzyme interact with lipid particles reported to be present in bovine seminal plasma. The purified PAF-AH, however, did display lipoprotein association properties in vitro similar to those demonstrated by the serum enzyme in vivo. At pH 7.4, it could associate with both low density lipoproteins and very low density lipoproteins but not with high density lipoproteins. Overall the data presented here indicate that the enzyme is strongly inactivated as a PAF-AH in seminal plasma and that it does not associate with lipid particles or spermatozoa.

NF-κB, a transcription factor, upregulates gene transcription of many inflammatory mediators. Here, we examined the activity of NF-κB in the rat small intestine, and how it may be affected by platelet-activating factor (PAF), an important mediator for intestinal injury and inflammation. Ileal nuclear extracts from sham-operated and PAF (1.5 μg/kg)-injected rats were prepared for the assessment of NF-κB DNA-binding activity, and the identification of NF-κB subunits. The experiment was also performed on neutrophil-depleted rats to examine whether the PAF effect is neutrophil-dependent. Cellular NF-κB was localized by immunohistochemistry. We found that: (a) NF-κB is constitutively active in rat small intestine; (b) PAF at a dose below that causing shock and bowel necrosis enhances DNA-binding activity of NF-κB within 30 min after injection; activated NF-κB contains predominantly p50 subunits; (c) immunohistochemistry showed that PAF induced translocation of p50 into the nucleus of cells of the lamina propria, as well as of the epithelium; and (d) the effect of PAF is abrogated by neutrophil depletion, suggesting a role of neutrophils in NF-κB activation. Our study suggests that NF-κB is weakly active constitutively in the intestine, and inflammatory stimuli such as PAF activate NF-κB and enhance its DNA-binding activity in the intestine, which contains predominantly p50 subunits.

Hypochlorous (HOCl) and hypobromous (HOBr) acids are strong oxidants derived from myeloperoxidase and eosinophil peroxidase, the major antimicrobial enzymes of neutrophils and eosinophils, respectively. These oxidants are highly reactive with a wide range of biomolecules. At physiological pH, both HOCl and HOBr react readily with amines to form haloamines and with the unsaturated bonds of fatty acids to form halohydrins. We have investigated which of these reactions occur with phosphatidylethanolamine (PE), the predominant phospholipid of Escherichia coli. The formation of haloamines was determined by TLC and colorimetrically and the formation of halohydrins was determined by TLC and GC-MS. With HOCl, chloramines were much the preferred product and chlorohydrins were formed in substantial amounts only when HOCl was in excess of the amount required to convert the amine to the dichloramine. With HOBr at all concentrations, bromamines and bromohydrins were formed concurrently, indicating a greater relative reactivity with unsaturated fatty acids than with HOCl. The bromamine derivatives of PE, and other primary amines, were found to be more reactive than the equivalent chloramines, and were able to brominate the unsaturated bonds of fatty acids. Bromohydrins (formed directly or through the action of bromamines) may, therefore, be suitable biomarkers for the production of HOBr in vivo.

The effects of long chain n-3 polyunsaturated fatty acids (PUFA) on the desaturation and elongation systems involved in the conversion of 18:3n-3 to 24:6n-3 were investigated. Microsomes were prepared from the livers of rainbow trout and incubated with ¹⁴C-labelled 18:3n-3 and cofactors required for elongation and/or desaturation in the presence of 22:6n-3, 24:5n-3 or 24:6n-3. The formation of 24:6n-3 was significantly inhibited in the presence of 50 μM 22:6n-3, 24:5n-3 or 24:6n-3, whereas the amount of radiolabelled 20:5n-3 formed was inhibited by only 24:5n-3 or 24:6n-3 at the same concentration. When malonyl-CoA was omitted from the incubation system to allow the measurement of desaturation in the absence of elongation, the Δ6 desaturation of ¹⁴C-18:3n-3 to ¹⁴C-18:4n-3 was inhibited by approximately 25% in the presence of 24:5n-3 or 24:6n-3 but was not affected by 22:6n-3. The Δ5 desaturation of ¹⁴C-20:4n-3 was not affected by the presence of any of the long chain PUFA and no significant effect of 18:3n-3, 22:6n-3 or 24:6n-3 on the Δ6 desaturation of 24:5n-3 to 24:6n-3 was observed. To permit the measurement of individual elongation reactions, KCN was included in the incubation medium to inhibit desaturation and ¹⁴C-labelled 18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3 and 22:5n-3 were examined as substrates. 18:4n-3 and 22:5n-3 were more extensively used for elongation than 18:3n-3, 20:4n-3 and 20:5n-3. The presence of 22:6n-3, 24:5n-3 or 24:6n-3 in the incubation system had no effect on any of the specific elongations of any of the substrates examined. It is concluded that, in the conversion of 18:3n-3 to 24:6n-3 by trout liver microsomes, the Δ6 desaturation of 18:3n-3 may be subjected to direct feedback inhibition and that 24:5n-3 may be preferred over 18:3n-3 as a substrate for Δ6 desaturation.