Overexpression, purification and characterization of human recombinant 15-lipoxygenase

Kühn, Hartmut; Barnett, Jim; Grünberger, D.; Baecker, Preston A.; Chow, Joan M.; Nguyen, Binh Q.; Bursztyn-Pettegrew, Hela; Chan, Hardy; Sigal, Elliott

doi:10.1016/0005-2760(93)90085-n

Cited by 129 publications

(98 citation statements)

References 25 publications

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“…Following amplification of the virus in Sf9 cells, viral stocks with titres of 1.4±2.5 Â 10 8 plaque forming units´mL 21 were generated for each of the splice variants. For protein expression, Hi5 cells were infected with recombinant baculovirus at an multiplicity of infection of 2 and the cells harvested after 72 h. Protein was partially purified by anion-exchange chromatography using methods previously described for 15-LOa [9].…”

Section: Baculovirus Expression Of the 15-lob Splice Variantsmentioning

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%

“…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.…”

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%

“…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].…”

mentioning

confidence: 99%

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

Kilty

Logan

Vickers

1999

European Journal of Biochemistry

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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...

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Section: Baculovirus Expression Of the 15-lob Splice Variantsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Kilty

Logan

Vickers

1999

European Journal of Biochemistry

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Structure conservation in lipoxygenases: Structural analysis of soybean lipoxygenase-1 and modeling of human lipoxygenases

et al. 1996

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Lipoxygenases are a class of non-heme iron dioxygenases which catalyze the hydroperoxidation of fatty acids for the biosynthesis of leukotrienes and lipoxins. The structure of the 839-residue soybean lipoxygenase-1 was used as a template to model human 5-, 12-, and 15-lipoxygenases. A distance-based algorithm for placing side chains in a low homology environment (only the four iron ligands were fixed during side chain placement) was devised. Twenty-six of the 56 conserved lipoxygenase residues were grouped in four distinct regions of the enzyme. These regions were analyzed to discern whether the side chain interactions could be duplicated in the models or whether alternate conformers should be considered. The effects of site directed mutagenesis variants were rationalized using the models of the human lipoxygenases. In particular, variants which shifted positional specificity between 12and 15-lipoxygenase activity were analyzed. Analysis of active site residues produced a model which accounts for observed lipoxygenase positional specificity and stereospecificity.

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Mass Spectrometry for Lipid Analyses

Mesaros

Blair

2008

Wiley Encyclopedia of Chemical Biology

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The increasing recognition that lipids have important biological functions in signal transduction in addition to their structural role in cell membranes has stimulated tremendous activity in the field of lipid research. Their complex structures and the potent biological activity of many individual lipids has necessitated the development of highly specific and sensitive methodology for their analysis. Mass spectrometry (MS)‐based methodology has become the technique of choice because of its ability to conduct analyses on trace amounts of bioactive lipids. Gas chromatography (GC)‐MS was widely used for the early studies on lipid analysis, but the applicability was limited. Development of the new atmospheric pressure ionization (API) techniques and matrix‐assisted laser desorption/ionization (MALDI) has significantly increased the range of lipids that can be analyzed by MS. Methodology based on these ionization techniques will continue to make an important contribution to lipid analysis for the foreseeable future.

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Overexpression, purification and characterization of human recombinant 15-lipoxygenase

Cited by 129 publications

References 25 publications

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

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

Structure conservation in lipoxygenases: Structural analysis of soybean lipoxygenase-1 and modeling of human lipoxygenases

Mass Spectrometry for Lipid Analyses

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