Native and mutant 5-lipoxygenase expression in a baculovirus/insect cell system.

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.

Section: Assays Of Purified 1zlipoxygenase Activitymentioning

confidence: 99%

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

Chen

Brash

Funk

1993

“…In vitro, both 5-LO and 15-LOa undergo a burst phase of activity before reaching a steady rate of product formation and then undergoing suicide inactivation by their respective enzyme products [9,14]. Investigations into the subcellular localization of both 5-LO and 15-LOa have demonstrated that these proteins become membrane associated in a calcium-dependent manner and upon membrane localization their specific activity is increased [16,17].…”

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confidence: 99%

“…For example, 12-HETE and 15-HETE have been implicated in tumour metastases and the formation of atherosclerotic plaques, respectively [11,12]. The important biological roles of these LO metabolites and the complexity of substrate utilization by members of the LO family emphasize the importance of understanding factors which affect the activity and regulation of these enzymes.Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis of these enzymes [9,13,14] and the hypotheses as to the mechanism by which LOs oxygenate fatty acids have been developed [15]. In vitro, both 5-LO and 15-LOa undergo a burst phase of activity before reaching a steady rate of product formation and then undergoing suicide inactivation by their respective enzyme products [9,14].…”

mentioning

confidence: 99%

“…Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis of these enzymes [9,13,14] and the hypotheses as to the mechanism by which LOs oxygenate fatty acids have been developed [15]. In vitro, both 5-LO and 15-LOa undergo a burst phase of activity before reaching a steady rate of product formation and then undergoing suicide inactivation by their respective enzyme products [9,14].…”

mentioning

confidence: 99%

See 1 more Smart Citation

Differential characteristics of human 15‐lipoxygenase isozymes and a novel splice variant of 15S‐lipoxygenase

Kilty

Logan

Vickers

1999

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5-LO, 12S-LO, 12R-LO, 15-LO and 15S-LO. Using partially purified recombinant 15S-LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15-LO. 15S-LO has a threefold higher K m , similar V max and increased specificity of oxygenation for arachidonic acid, and a similar K m but decreased V max for linoleic acid in comparison to 15-LO. Unlike 15-LO, 15S-LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S-LO activity is regulated through calcium-dependent association of the enzyme with the membrane fraction of cells.In addition, whilst independently cloning the recently described 15S-LO, we identified a splice variant containing an in-frame 87-bp deletion corresponding to amino acids 401±429 inclusive. Modelling of the 15S-LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete a-helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates.Alternative splicing of 15S-LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15-LO isozymes which may reflect differing roles for the proteins in vivo. Although the LOs produce specific hydroxyeicosatetraenoic (HETE) enantiomers, the enzymes do not all display absolute positional specificity with respect to oxygenation of AA. For example, while 15-LOb exclusively oxygenates C15 of AA [8], 15-LOa oxygenates AA at both C15 and C12 [9]. In addition, the degree of homology between members of the LO family does not define the product profile. For example, amino acid sequence alignments demonstrate that 15-LOb is more closely related to 12R-LO than 15-LOa, suggesting complex evolutionary paths in the LO family.The LOs are responsible for the synthesis of a number of inflammatory mediators, including leukotrienes and lipoxins, which are implicated in inflammatory disorders such as bronchial asthma [10]. Lipoxygenase metabolites have also been implicated in a number of noninflammatory biological processes. For example, 12-HETE and 15-HETE have been implicated in tumour metastases and the formation of atherosclerotic plaques, respectively [11,12]. The important biological roles of these LO metabolites and the complexity of substrate utilization by members of the LO family emphasize the importance of understanding factors which affect the activity and regulation of these enzymes.Overexpression and characterization of recombinant 5-LO, 12-LO and 15-LOa have been carried out allowing kinetic analysis...

“…Five of these histidine residues have been suggested by Shibata et al [19] to be putative iron-binding sites. Funk et al [27], however, reported that site-directed mutation of His362 and His372 within the putative iron-binding domain of human 5-lipoxygenase did not alter the enzyme activity, suggesting that the two residues are not involved in iron binding. Thus, the function of these conserved residues still remains unclear.…”

Section: Discussionmentioning

confidence: 99%

cDNA cloning of rice lipoxygenase L‐2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli

Ohta

Shirano

Tanaka

et al. 1992

A full-length cDNA of rice lipoxygenase L-2 was cloned from 3-day-old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15°C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons (93% G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was characterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and K,. The enzyme had the same properties as the L-2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L-3 isolated from mature plants.Lipoxygenase catalyzes hydroperoxidation of fatty acids by the introduction of molecular oxygen onto the cis, cis-1,4-pentadiene structure of its substrates. The hydroperoxy fatty acids are then metabolized into physiologically active compounds. Eicosanoids, which are derived from arachidonic acid via lipoxygenation pathways in mammalian cells have been extensively investigated [ 11. Jasmonic acid, derived from linolenic acid via lipoxygenation in plant cells, is recognized as a gaseous growth regulator [2]. Extensive enzyme studies of lipoxygenases, especially soybean lipoxygenase L-1 , have been carried out [3] and two recent reviews of plant lipoxygenases have been published [4, 51. Recently, biosynthesis of anti-fungal compounds via a lipoxygenase pathway was proposed by Ohta et al. [6]. The same authors also demonstrated an increase in lipoxygenase activity in rice leaves in response to infection with an incompatible strain of blast fungus [7] (a compatible strain did not elicit increased lipoxygenase activity), suggesting an important role for lipoxygenase in plant disease resistance.Characterization of rice lipoxygenases in mature seeds has been reported [8 -101. The seeds contain three distinct lipoxygenases, L-1, L-2 and L-3. They differ from each other not only in their enzymic properties, but also in their physiological aspects. The L-3 enzyme is the major lipoxygenase in Correspondence to D. Shibata, Mitsui Plant Biotechnology Research Institute, TCI-AIO, Sengen 2-1-6, Tsukuba, Ibaraki, Japan 305Abbreviation. L-E, an active enzyme expressed from the full-length cDNA encoding a rice lipoxygenase in E. coli.Enzyme. Lipoxygenase (EC 1.13.11.12).Note. The nucleotide-sequence data published here have been submitted to the EMBL sequence data bank and are available under acces...