Characterization of human 12-lipoxygenase genes.

Funk, Colin D.; Funk, Lena B.; FitzGerald, Garret A.; Samuelsson, Bengt

doi:10.1073/pnas.89.9.3962

Cited by 85 publications

(31 citation statements)

References 39 publications

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“…This relatively low amount of protein correlates with the low abundance of 12-lipoxygenase mRNA in HEL cells [12]. Previously, 'platelet-type' 12-lipoxygenase mRNA had been detected in HUVE cells by reverse-&anscription/PCR analysis [28]; however, we were unable to detect 12-lipoxygenase protein by immunoblot or immunolabeling experiments here. Therefore, there is still no convincing evidence for expression of the platelet form of 12-lipoxygenase outside of platelets or platelet-like cell lines (HEL and DAMI)[4-6, 12, 291.…”

Section: Discussioncontrasting

confidence: 50%

Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

Chen

Brash

Funk

1993

European Journal of Biochemistry

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A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 1Zlipoxygenase activity and protein levels were achieved ; the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His),-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetraenoic acid with little, if any, epoxyalcohol or reduction products and had a V,,, of 2-4 pmol min-' mg protein-', K , of 10 pM and k,,, of =250 min-I. Linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same K,. The enzyme was most active between pH 7.5 -8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-lipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1 % of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-lipoxygenase should facilitate detailed biochemical structure/function studies.Arachidonate 12-lipoxygenase catalyzes the insertion of molecular oxygen stereospecifically onto C-12 of arachidonic acid to yield (1 2S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) [l-31. This was the first described mammalian lipoxygenase, found in platelets of different species [l, 31. More recently, the cDNA encoding human platelet 12-lipoxygenase has been isolated [4-61. The enzyme consists of 663 amino acids with a molecular mass of 75 kDa. Human and bovine platelet 12-lipoxygenases differ distinctly from the 12-lipoxygenases found in porcine and bovine leukocytes by several biochemical and immunological criteria [7, 81. The porcine leukocyte enzyme has been purified and characterized extensively [9]. This enzyme is 65% identical to the human platelet enzyme based on the deduced primary structure [4][5][6] 101. Although the human Correspondence to C.

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Section: Discussioncontrasting

confidence: 50%

Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

Chen

Brash

Funk

1993

European Journal of Biochemistry

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“…1 and Table 2). The gene encoding 5-LO (ALOX5) spans 82 kb on 10q11.2 (65). The 85 kDa protein encoded is composed of a 673 amino acid polypeptide (66).…”

Section: Lt Synthesismentioning

confidence: 99%

Leukotriene pathway genetics and pharmacogenetics in allergy

Duroudier

Tulah

Sayers

2009

Allergy

102

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Leukotrienes (LT) are a family of potent eicosanoid lipid mediators with central importance in disease processes such as inflammation and proliferation most notably observed in allergic conditions like asthma (1). There are two general classes of LT according to the presence of a cysteine residue in their amino acid chain: the cysteinyl leukotrienes (CysLTs) including LTC 4 , LTD 4 and LTE 4 and the dihydroxyleukotriene LTB 4 (2). The 5-lipoxygenase pathwayLeukotrienes are produced in a multi-step enzyme pathway called the 5-lipoxygenase (5-LO) pathway, which is active in leucocytes such as neutrophils, eosinophils, mast cells and monocytes (Fig. 1). Arachidonic acid is the precursor for LT synthesis and is hydrolysed from the plasma membrane by cytosolic phospholipase A 2 (and other isoforms) (3, 4) in a calcium-dependent process (5). Upon activation, the rate-limiting enzyme 5-LO oxygenates first free arachidonic acid into the unstable intermediate 5-hydroperoxyeicosatetraenoic acid (5-HPETE) which is then either hydrolysed to 5-hydroxyeicosatetraenoic acid (5-HETE) or transformed into the unstable epoxide leukotriene A 4 (LTA 4 ) by forming a conjugated triene system through dehydration (6). 5-LO translocates from either the nucleus (in macrophages) or cyotosol (in neutrophils) to the nuclear envelope in response to cell activation (7,8). This movement occurs as 5-LO is dependent on a 5-LO activating protein (FLAP) for its function. FLAP remains associated with the nuclear membrane (9) and acts as a Ôtransfer proteinÕ presenting arachidonic acid to 5-LO, a system which allows the favourable conversion of 5-HPETE to LTA 4 compared to 5-HETE (10). Both 5-LO and FLAP are expressed in cells of myeloid lineage (11) restricting the pathway to these cell types. LTA 4 can be further metabolised to the cysteinyl leukotrienes (CysLTs) or LTB 4 . The specific glutathione S-transferase leukotriene C 4 synthase (LTC 4 S) conjugates LTA 4 to form LTC 4 which can then be rapidly converted to LTD 4 by a gammaglutamyl transpeptidase and to LTE 4 by a dipeptidase once exported out the cell by membrane transport proteins such as multi-drug related protein 1 (MRP1) (12-15). A specific zinc metallohydrolase, LTA 4 hydrolase (LTA 4 H) is responsible for the conversion of LTA 4 to LTB 4 (16). CysLTs and LTB 4 act on different G-protein coupled receptors (GPCRs). Each bind to at least two different receptors: CysLTs to CYSLTR1 and CYSLTR2 and LTB 4 to LTB 4 R1 and LTB 4 R2 (or BLT1 and BLT2) (17).Leukotrienes (LT) are biologically active lipid mediators known to be involved in allergic inflammation. Leukotrienes have been shown to mediate diverse features of allergic conditions including inflammatory cell chemotaxis/activation and smooth muscle contraction. Cysteinyl leukotrienes (LTC 4 , LTD 4 and, LTE 4 ) and the dihydroxy leukotriene LTB 4 are generated by a series of enzymes/ proteins constituting the LT synthetic pathway or 5-lipoxygenase (5-LO) pathway. Their function is mediated by interacting with multiple receptors. Leukotr...

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“…In contrast, the leukocyte 12-LO (lk12-LO) is more promiscuous and shares significant sequence similarity and substrate specificity with 15-LO-1. Though two 12S-LO genes have been characterized in humans, these correspond to the pl12-LO and a pseudogene thought to arise from gene duplication [13]. It is predicted that 15-LO-1 is the human homologue of lk12-LO [9].…”

Section: Introductionmentioning

confidence: 99%

Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases

Prusakiewicz¹,

Turman²,

Vila³

et al. 2007

Archives of Biochemistry and Biophysics

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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 lipoaminoacids or vanilloids or may convert them to bioactive derivatives.

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Characterization of human 12-lipoxygenase genes.

Cited by 85 publications

References 39 publications

Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

Purification and characterization of recombinant histidine‐tagged human platelet 12‐lipoxygenase expressed in a baculovirus/insect cell system

Leukotriene pathway genetics and pharmacogenetics in allergy

Oxidative metabolism of lipoamino acids and vanilloids by lipoxygenases and cyclooxygenases

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