Publications

• Cloning and expression of an airway epithelial 12-lipoxygenase.

  de Marzo N., Sloan D.L., Dicharry S., Highland E., Sigal E.

  Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9- ... >> More

  Arachidonate 12-lipoxygenase generates metabolites that may regulate airway function. To further characterize this enzyme, we isolated a cDNA corresponding to 12-lipoxygenase from a bovine tracheal epithelium cDNA library using human reticulocyte 15-lipoxygenase cDNA as a probe. The resulting 2.9-kb cDNA, the identity of which was confirmed by expression of active catalytic function in Escherichia coli has a 2.0-kb open reading frame encoding a protein of 75,000 kDa and includes 5 bp of 5'-untranslated region and 0.9 kb of 3'-untranslated region. On Northern blots, the 12-lipoxygenase cDNA hybridized to one band (3.5 kb) of bovine tracheal epithelium RNA. Polyclonal antibodies that recognize human tracheal 15-lipoxygenase cross-reacted on immunoblots to the expressed bovine tracheal 12-lipoxygenase. Further, the deduced amino acid sequence is 86% identical (93% similar) to human 15-lipoxygenase but 64% identical to human platelet 12-lipoxygenase, suggesting that the bovine tracheal enzyme is the homologue of the human 15-lipoxygenase. This is the first sequence of an epithelial lipoxygenase from any species. A comparison of the bovine sequence with other lipoxygenase sequences shows that there are only four amino acids which are conserved differences between a 12-lipoxygenase and a 15-lipoxygenase. We hypothesize that these four amino acids may be responsible for the positional specificity of the enzyme. << Less

  Am. J. Physiol. 262:L198-L207(1992) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase.

  Suzuki H., Kishimoto K., Yoshimoto T., Yamamoto S., Kanai F., Ebina Y., Miyatake A., Tanabe T.

  cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which w ... >> More

  cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaffinity-purified to near homogeneity. Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase. Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7. << Less

  Biochim. Biophys. Acta 1210:308-316(1994) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enzymic characterization of epidermis-derived 12-lipoxygenase isoenzymes.

  Siebert M., Krieg P., Lehmann W.D., Marks F., Fuerstenberger G.

  Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactiv ... >> More

  Substrate selectivity and other enzymic characteristics of two epidermis-derived lipoxygenases (LOXs), the epidermis-type (e) (12S)-LOX and (12R)-LOX, were compared with those of the platelet-type (p) (12S)-LOX. In contrast with p(12S)-LOX, e(12S)-LOX and (12R)-LOX exhibited no or very low reactivity towards the customary substrates linoleic acid and arachidonic acid but metabolized the corresponding fatty acid methyl esters, which, in contrast, were not accepted as substrates by p(12S)-LOX. Other esters of arachidonic acid and linoleic acid, including propan-2-yl and cholesterol esters, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-linoleyl-sn-glycero-3-phosphoethanolamine, and ceramide 1 carrying an omega-linoleic acid ester, were not metabolized by these three LOX isoenzymes. Among various polyunsaturated fatty acids the isomeric eicosatrienoic acids were found to be oxygenated by e(12S)-LOX but not by (12R)-LOX. 4,7,10,13,16,19-Docosahexaenoic acid as a substrate was restricted to p(12S)-LOX. Variations in the pH and the Ca(2+) content of the incubation medium affected the catalytic potential only slightly. Whereas (12R)-LOX activity increased in the presence of Ca(2+) and with an acidic pH, Ca(2+) had no effect on p(12S)-LOX and e(12S)-LOX; an acidic pH decreased the catalytic activity of the latter two. However, the catalytic activity of the epidermis-type isoenzymes, but not of p(12S)-LOX, was found to be markedly increased in the presence of DMSO. Under these conditions, e(12S)-LOX and (12R)-LOX oxygenated 4,7,10,13,16,19-docosahexaenoic acid to 14-hydroxy-4,7,10,12,16,19-docosahexaenoic acid and 13-hydroxy-4,7,10,14,16,19-docosahexaenoic acid respectively. In addition, (9R)-hydroxyoctadeca-10,12-dienoic acid methyl ester was generated from linoleic acid methyl ester by (12R)-LOX. Independently of the substrate, the catalytic activity of e(12S)-LOX and (12R)-LOX was always at most 2% of that of p(12S)-LOX with arachidonic acid as substrate. << Less

  Biochem. J. 355:97-104(2001) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Systematic analysis of rat 12/15-lipoxygenase enzymes reveals critical role for spinal eLOX3 hepoxilin synthase activity in inflammatory hyperalgesia.

  Gregus A.M., Dumlao D.S., Wei S.C., Norris P.C., Catella L.C., Meyerstein F.G., Buczynski M.W., Steinauer J.J., Fitzsimmons B.L., Yaksh T.L., Dennis E.A.

  Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we ove ... >> More

  Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 μM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors. << Less

  FASEB J. 27:1939-1949(2013) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases.

  Chen X.-S., Kurre U., Jenkins N.A., Copeland N.G., Funk C.D.

  Two types of 12-lipoxygenase that catalyze the transformation of arachidonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories. Here, we document, for the first time, a molecular characterization of both forms ... >> More

  Two types of 12-lipoxygenase that catalyze the transformation of arachidonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories. Here, we document, for the first time, a molecular characterization of both forms within the same species. The amino acid sequence of the murine platelet 12-lipoxygenase deduced from its cDNA is 58% identical to the murine spleen/leukocyte 12-lipoxygenase. Expression constructs carrying the cDNAs for the two 12-lipoxygenase forms were introduced into human embryonic kidney 293 cells. The platelet-type enzyme metabolized arachidonic acid exclusively to 12-HPETE, whereas the leukocyte-type enzyme formed both 12-HPETE and 15-hydro(pero)xyeicosatetraenoic acid in a ratio of approximately 3:1. Linoleic acid was metabolized to a similar extent by the latter enzyme to 13-hydro(pero)xyoctadecadienoic acid but not by the platelet enzyme. Mutagenesis and deletion of the highly conserved lipoxygenase C-terminal isoleucine (Ile663), a residue believed to be involved in the non-heme iron atom coordination of all lipoxygenases, was performed. Deletion of Ile663 and substitution with most amino acids abolished enzyme activity. Only a valine substitution retained significant activity. These findings would tend to indicate a stringent requirement for the proper spatial alignment and folding of the C-terminal chain back into the core of the enzyme to interact with the iron atom by analogy with the recently determined crystal structure of a soybean lipoxygenase (Boyington, J. C., Gaffney, B. J., and Amzel, L. M. (1993) Science 260, 1482-1486). The platelet-type and leukocyte-type 12-lipoxygenase genes were cloned from a murine 129 Sv genomic library. Both genes are divided into a similar 14-exon/13-intron format, with the platelet-type gene being approximately twice the size of the leukocyte-type gene (13 versus 7.5 kilobases). A segment of a third gene was also isolated and probably represents a pseudogene derivative of either of these 12-lipoxygenase genes. All three genes were mapped to the central region of mouse chromosome 11 in a region of homology with human chromosome 17. Antibodies prepared against the two forms of 12-lipoxygenase revealed the differential distribution of the two enzymes throughout the mouse. << Less

  J. Biol. Chem. 269:13979-13987(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation.

  Ikei K.N., Yeung J., Apopa P.L., Ceja J., Vesci J., Holman T.R., Holinstat M.

  Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can b ... >> More

  Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane. << Less

  J. Lipid Res. 53:2546-2559(2012) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases.

  Prusakiewicz J.J., Turman M.V., Vila A., Ball H.L., Al-Mestarihi A.H., Di Marzo V., Marnett L.J.

  The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of ... >> More

  The lipoamino acids and endovanilloids have multiple roles in nociception, pain, and inflammation, yet their biological reactivity has not been fully characterized. Cyclooxygenases (COXs) and lipoxygenases (LOs) oxygenate polyunsaturated fatty acids to generate signaling molecules. The ability of COXs and LOs to oxygenate arachidonyl-derived lipoamino acids and vanilloids was investigated. COX-1 and COX-2 were able to minimally metabolize many of these species. However, the lipoamino acids were efficiently oxygenated by 12S- and 15S-LOs. The kinetics and products of oxygenation by LOs were characterized. Whereas 15S-LOs retained positional specificity of oxygenation with these novel substrates, platelet-type 12S-LO acted as a 12/15-LO. Fatty acid oxygenases may play an important role in the metabolic inactivation of lipoamino acids or vanilloids or may convert them to bioactive derivatives. << Less

  Arch. Biochem. Biophys. 464:260-268(2007) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.