Calcium stimulation of a novel 12-lipoxygenase from rat basophilic leukemia (RBL-1) cells

Cytosolic fraction of RBL-1 cells transformed arachidonic acid to 12-HETE in addition to the well-recognized 5-hydroxyeicosatetraenoic acid (5-HETE) in the presence of Ca2+, Mg2+ or Mn2+. The identity of 12-HETE was confirmed by gas chromatography-mass spectrometry. Syntheses of 12-HETE and 5-HETE were catalyzed by separate lipoxygenases, since the formation of each product showed differential sensitivity to inhibitors and temperature. 12-Lipoxygenase from RBL-1 cells was also found to be distinct from the enzyme from platelets in calcium sensitivity.     

Tetrapetalone A, a novel lipoxygenase inhibitor from Streptomyces sp

A simple new assay was designed for lipoxygenase inhibitors. This assay was used to find the novel lipoxygenase inhibitor, tetrapetalone A (1). Tetrapetalone A (1), C26H33NO7, was isolated from Streptomyces sp. USF-4727 strain. Its planar structure was determined by spectroscopic evidence and by methylating with diazomethane to show the presence of a novel tetracyclic skeleton and a beta-D-rhodinosyl moiety. The stereochemistry of 1 was investigated by the coupling constant in the 1H-NMR spectrum, NOE correlations, modified Mosher's method and derivation. We have reported the structural elucidation of 1 in our previous paper. However, further investigation gave another structure for 1, which is described in this paper. Tetrapetalone A showed similar inhibitory activity against soybean lipoxygenase to the two well-known lipoxygenase inhibitors, kojic acid and NDGA, while methylated tetrapetalone A (2) showed little inhibitory activity, even at a concentration of 1 mM.     

Purification of a novel lipoxygenase from eggplant (Solanum melongena) fruit chloroplasts

A novel membrane lipoxygenase (LOX; EC 1.13.11.12) from eggplant ( Solanum melongena L. cv. Belleza negra) fruit chloroplasts has been purified 20-fold to a specific activity of 207 enzymatic units per mg of protein with a yield of 72%. The purification was carried out by sonicating the chloroplastic membranes in the presence of Triton X-114 followed by phase partitioning and anion exchange chromatography. The purified membrane LOX preparation consisted of a single major band with an apparent molecular mass of 97 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results obtained using intact chloroplasts indicate that the enzyme is not localized in the stroma. When the enzyme reacts with linoleic acid, it produces a single peak, which comigrates with standard 9-hydroperoxy-octadecadienoic acid. A physiological role for this chloroplastic LOX is proposed.     

Bioactive lipoxygenase metabolites stimulation of NADPH oxidases and reactive oxygen species

In mammalian cells, reactive oxygen species (ROS) are produced via a variety of cellular oxidative processes, including the activity of NADPH oxidases (NOX), the activity of xanthine oxidases, the metabolism of arachidonic acid (AA) by lipoxygenases (LOX) and cyclooxygenases (COX), and the mitochondrial respiratory chain. Although NOX-generated ROS are the best characterized examples of ROS in mammalian cells, ROS are also generated by the oxidative metabolism (e.g., via LOX and COX) of AA that is released from the membrane phospholipids via the activity of cytosolic phospholipase A₂ (cPLA₂). Recently, growing evidence suggests that LOX- and COX-generated AA metabolites can induce ROS generation by stimulating NOX and that a potential signaling connection exits between the LOX/COX metabolites and NOX. In this review, we discuss the results of recent studies that report the generation of ROS by LOX metabolites, especially 5-LOX metabolites, via NOX stimulation. In particular, we have focused on the contribution of leukotriene B₄ (LTB₄), a potent bioactive eicosanoid that is derived from 5-LOX, and its receptors, BLT1 and BLT2, to NOX stimulation through a signaling mechanism that leads to ROS generation.     

Calcium stimulation of polyphenylalanine synthesis in a bovine heart cell-free system

A bovine myocardial cell-free system, active in polyphenylalanine synthesis, has been studied. When Ca²⁺ was present under suboptimal Mg²⁺ concentrations (2 and 5 mM) a marked stimulation of the poly(U) directed macromolecular synthesis was obtained. Calcium did not stimulate the aminoacylation of bovine heart tRNA^Phe The evidence suggests that calcium is required, in conjunction with other cations, for an efficient translation of synthetic polynucleotides.     

Regiospecificity of a novel bacterial lipoxygenase from Myxococcus xanthus for polyunsaturated fatty acids

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific?oxygenated signaling compounds.     

TNF induces c-fos via a novel pathway requiring conversion of arachidonic acid to a lipoxygenase metabolite.

Tumour necrosis factor (TNF), a lymphokine released by activated macrophages, has diverse effects on a wide variety of cell types. TNF exerts these effects via specific cell surface receptors; however little is known of the biochemical events that ensue. We have shown that TNF rapidly induces the proto-oncogenes c-fos and c-jun in the adipogenic TA1 cell line and have used these responses to characterize the intracellular mediators of TNF action. We find that arachidonic acid, which is released in response to TNF, induces c-fos, but not c-jun mRNA in quiescent TA1 cells. Pretreatment of the cells with lipoxygenase inhibitors abolishes the induction of c-fos by TNF, while the induction of c-jun is unaffected; in contrast, a cyclooxygenase inhibitor has no effect on either response. Finally, we have demonstrated that TNF stimulates production of lipoxygenase metabolites in TA1 cells and that one of these, 5-HPETE, induces c-fos, but not c-jun. These data suggest that TNF activates two second messenger pathways, one of which is dependent on release of arachidonic acid and its subsequent conversion to a lipoxygenase metabolite.     

Calcium ion influx during mitogenic stimulation of lymphocytes

The uptake of free calcium ion (Ca2+) in PHA- or A23187-stimulated lymphocytes was measured using 45CaCl2 and 3H-water. Augmentation of Ca2+ uptake by both mitogens was observed, but the enhanced uptake occurred transiently, sometime within 30 min of the stimulation. The total amount of calcium in quiescent lymphocytes as determined by atomic absorption spectroscopy was about 2.9 X 10(-15) g/cell. When stimulated with PHA, more calcium gradually accumulated in the cells. The maximum amount of accumulation occurred at around 40 h, and was about 2-fold higher than that of control cells. In A23187-stimulated cells, the calcium content increased within 1 h by about 4-fold, reached a maximum at about 6 h (6-fold) and thereafter, surplus calcium was pumped out. The cytosolic free calcium ion concentration (the [Ca2+]i) within single cells was measured using quin 2 or fura-2. The [Ca2+]i was about 1 X 10(-7) M, and a transient increase in the [Ca2+]i was observed in some cells within 1 min after Con A-stimulation. Another rise in the [Ca2+]i was observed around the 40th h, and the maximum expression of the IL-2 receptor was observed at about this time. Therefore the results may indicate that the IL-2-mediated lymphocyte transformation is dependent on the rise in the [Ca2+]i.     

cDNA cloning, genomic structure, and chromosomal localization of a novel murine epidermis-type lipoxygenase.

Using a combination of degenerate PCR technique and conventional screening procedures, we isolated a cDNA encoding a novel lipoxygenase, termed epidermis-type lipoxygenase-3 (e-LOX-3, gene symbol Aloxe3), from mouse skin. Aloxe3 mRNA is expressed in the stratified epithelia of skin, tongue, and forestomach. The cDNA encodes a protein of 711 amino acids with a calculated molecular mass of 80.6 kDa. The amino acid sequence shows approximately 54% identity to the recently identified 12(R)-lipoxygenase. Sequence comparison revealed a segment of 41 amino acid residues localized near the boundary between the N- and the C-terminal domain sequences of the molecule, a structural feature that is also characteristic of 12(R)-lipoxygenase, suggesting that these two epidermis-derived lipoxygenases may be members of a novel structural class of mammalian lipoxygenases. The novel lipoxygenase gene is divided into 15 exons and 14 introns, spanning 22.3 kb of genomic DNA. By interspecific backcross analysis, the novel gene was localized to the central region of mouse chromosome 11.     

Crystal structure of a lipoxygenase from Cyanothece sp. may reveal novel features for substrate acquisition

In eukaryotes, oxidized PUFAs, so-called oxylipins, are vital signaling molecules. The first step in their biosynthesis may be catalyzed by a lipoxygenase (LOX), which forms hydroperoxides by introducing dioxygen into PUFAs. Here we characterized CspLOX1, a phylogenetically distant LOX family member from Cyanothece sp. PCC 8801 and determined its crystal structure. In addition to the classical two domains found in plant, animal, and coral LOXs, we identified an N-terminal helical extension, reminiscent of the long α-helical insertion in Pseudomonas aeruginosa LOX. In liposome flotation studies, this helical extension, rather than the β-barrel domain, was crucial for a membrane binding function. Additionally, CspLOX1 could oxygenate 1,2-diarachidonyl-sn-glycero-3-phosphocholine, suggesting that the enzyme may act directly on membranes and that fatty acids bind to the active site in a tail-first orientation. This binding mode is further supported by the fact that CspLOX1 catalyzed oxygenation at the n-10 position of both linoleic and arachidonic acid, resulting in 9R- and 11R-hydroperoxides, respectively. Together these results reveal unifying structural features of LOXs and their function. While the core of the active site is important for lipoxygenation and thus highly conserved, peripheral domains functioning in membrane and substrate binding are more variable.     

Expression profiles of two novel lipoxygenase genes in Populus deltoides

In the present study, we cloned two lipoxygenase genes, PdLOX1 and PdLOX2 (GenBank accession no. DQ131178, DQ131179), from Populus deltoides cv. ‘Lux’ (I-69/55). A prokaryotic expression analysis of PdLOX1 and PdLOX2 revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the expected lipoxygenase activities. Chromatogram analysis indicated that the two lipoxygenase mainly possess 13-LOX activity. Phylogenetic analysis of the derived amino acid sequences of known lipoxygenases revealed that PdLOX1 and PdLOX2 were members of the type 2 13-LOX family of genes. This class of lipoxygenases is known to be involved in biotic and abiotic stress. Using real-time RT-PCR, we evaluated PdLOX1 and PdLOX2 expression following exposure to a Poplar fungal pathogen (Marssonina brunnea f. sp. Multigermtubi), mechanical injury, methyl jasmonate (MeJA), or salicylic acid (SA). We report that both PdLOX1 and PdLOX2 expression levels were increased following exposure to M. brunnea f. sp. Multigermtubi, with the pathogen exerting a relatively stronger influence on PdLOX1 expression. Furthermore, expression levels of the two genes were also up-regulated by mechanical damage and exposure to MeJA. In contrast, both PdLOX1 and PdLOX2 expression was down-regulated by SA treatment. We propose that the two novel lipoxygenases may play an important role in Poplar resistance to biotic and abiotic stress.     

Calcium lonophore-induced stimulation of secretory activity in Chlamydomonas reinhardii

The cell-wall lysin in gametes from Chlamydomonas reinhardii which under normal mating conditions is activated by flagellar cell contact was found to be susceptible to stimulation by the antibiotic ionophore A 23187 provided that CA²⁺ was included in the medium. Ionophore-induced release of the cell-wall lysin did not deend on the mating type or the gametic state of the cells. Vegetative cells which normally do not exhibit any mating capacity reacted with cell-wall lysis like gametes stimulated by cell contact.Ionophore-dependent Ca²⁺-transfer across the cell membranes generated a signal for cell-wall lysis only in cells with intact flagella. Deflagellated cells did not respond to A 23187 before regeneration of the amputated organelles. Another indication for a possible role of flagella in Ca²⁺-mediated cell-wall lysis was obtained from a conditional flagellar-assembly mutant of C. reinhardii which had been isolated and described by Huang et al. (1977). Upon shift-up the mutant strain immediately became unresponsive to A 23187 and Ca²⁺ but regained susceptibility soon after being retransferred to permissive conditions (20°C).     

An ensemble of lipoxygenase structures reveals novel conformations of the Fe coordination sphere

The regio‐ and stereo‐specific oxygenation of polyunsaturated fatty acids is catalyzed by lipoxygenases (LOX); both Fe and Mn forms of the enzyme have been described. Structural elements of the Fe and Mn coordination spheres and the helical catalytic domain in which the metal center resides are highly conserved. However, animal, plant, and microbial LOX each have distinct features. We report five crystal structures of a LOX from the fungal plant pathogen Fusarium graminearum. This LOX displays a novel amino terminal extension that provides a wrapping domain for dimerization. Moreover, this extension appears to interfere with the iron coordination sphere, as the typical LOX configuration is not observed at the catalytic metal when the enzyme is dimeric. Instead novel tetra‐, penta‐, and hexa‐coordinate Fe²⁺ ligations are apparent. In contrast, a monomeric structure indicates that with repositioning of the amino terminal segment, the enzyme can assume a productive conformation with the canonical Fe²⁺ coordination sphere.     

Arachidonic acid metabolism in rat pancreatic acinar cells: calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromatography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8-10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Arachidonic acid metabolim in rat pancreatic acinar cells: Calcium-mediated stimulation of the lipoxygenase system

Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [¹⁴C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromtography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8–10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonic derivative was stimulated by the calcium ionophore ionomycin. Stimulation of lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF_1α, PGF_2α, and PGE₂ were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulates the lipoxygenase pathway, the precise role of these arachidonate metabolites in the physiology of the exocrine pancreas is still obscure.     

Cloning of a rabbit erythroid-cell-specific lipoxygenase mRNA

We report the isolation of cDNA recombinants representing part of the rabbit reticulocyte (immature red blood cell, RBC) lipoxygenase (LOX) mRNA. One cDNA predicts an amino acid (aa) sequence matching exactly the unique N-terminal 30-aa sequence of the purified enzyme. Further, the reticulocyte mRNA, hybrid-selected by this recombinant, can be translated in vitro to give a polypeptide that comigrates with the purified reticulocyte LOX and is recognized by affinity-purified anti-RBC LOX polyclonal antibodies. Southern blotting experiments hybridising the RBC LOX cDNAs available to total rabbit genomic DNA digested with various restriction enzymes gives a fairly simple hybridisation pattern under moderate stringency conditions: moreover, the same pattern is obtained with a cloned fragment of genomic DNA containing the RBC LOX gene. This indicates that the RBC LOX gene is unique in the genome and seems not to be very closely related to the genes encoding the other tissue LOXs. We also show by Northern transfer/hybridisation experiments that the RBC LOX mRNA is expressed only in the red cell lineage but not in white blood cells (bone marrow or spleen) or in other non-erythroid cells tested (e.g., brain and lung).     

Analysis of a lipoxygenase pseudogene in Pisum

 Lipoxygenase (LOX) enzymes play important roles in plant biology, and in the quality of plant-derived foods, through the production of fatty acid hydroperoxides that are metabolized either to jasmonate, or to volatile aldehydes that are part of plant defence systems and/or impart tastes and aromas to fruits and vegetables. We have identified a lipoxygenase pseudogene in peas that is composed of three elements: rearranged LOX-2 and LOX-3 genes and an unidentified tract of DNA. We present evidence that such an arrangement is normally present in the genome of Pisum sativum, but is absent from Pisum fulvum lines, including a mutant line that lacks LOX-2 polypeptides. The absence of LOX-2 polypeptides and the pseudogene co-segregate. The pseudogene therefore has utility as a molecular marker for the introgression of the LOX-2-null phenotype into commercial Pisum sativum genotypes. Received: 27 April 1998 / Accepted: 17 September 1998     

Calcium stimulation of glutamine hydrolysis in synaptosomes from rat brain

Calcium stimulates the hydrolysis of glutamine in synaptosomes prepared from rat brain both by the sucrose- (12) and the Ficoll/sucrose-gradient techniques (13). The calcium activation is phosphate-dependent and maximal effect is obtained at a calcium concentration of 0.5-1.0 mM. It is reduced by increasing the numbers of synaptosomes in the incubation mixture, and abolished by the product inhibitors of glutaminase, glutamate and ammonia, but unaffected by the uncoupler 2,4-dinitrophenol which inhibits the mitochondrial proton pump. Moreover, since the hydrolysis of glutamine is mediated by glutaminase (EC 3.5.1.2), and calcium does not activate the purified enzyme, an indirect phosphate-dependent effect of calcium on glutaminase is most likely. Calcium activates preferentially the N-ethylmaleimide insensitive fraction of glutaminase. The calcium activation is not dependent on synaptosomal membranes as it is found in synaptosomes subject to previous freezing. It is also found in isolated synaptosomal mitochondria and is thus a property of nerve endings. The calcium activation of glutaminase is unaffected by potassium in depolarizing concentrations, and may not be directly involved in the neurotransmission processes, but possibly in replenishing depleted stores of transmitter glutamate.