12-hydroxy-5-8-10-14-eicosatetraenoic-acid and nickel-nitrilotriacetic-acid
12-hydroxy-5-8-10-14-eicosatetraenoic-acid has been researched along with nickel-nitrilotriacetic-acid* in 1 studies
Other Studies
1 other study(ies) available for 12-hydroxy-5-8-10-14-eicosatetraenoic-acid and nickel-nitrilotriacetic-acid
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Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization.
Arachidonate 12-lipoxygenase (12-LO) from porcine leukocytes was expressed in insect cells using a baculovirus expression vector. The recombinant 12-LO was expressed as an N-terminal fusion protein with a 31-amino acid polypeptide carrying a six-histidine tag and an enterokinase cleavage site. Maximal intracellular enzyme activity and protein levels were observed 48 h after infection of Spodoptera frugiperda cells with the recombinant virus. Cells were lysed and the recombinant protein was purified in a single step by Ni2+-nitrilotriacetate column chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. Recombinant enzyme catalyzed the formation of 12-hydroperoxy-5,8,10,14-eicosatetranoic acid and a small amount of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Chiral-phase HPLC analysis indicated that the 12-(S) enantiomer was the predominant product. The purified recombinant 12-lipoxygenase oxygenated linoleic acid to about 19% of the extent of oxygenation of arachidonic acid. Nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid inhibited the recombinant enzyme with IC50's of 2.2 and 0.06 micM, respectively. Expression of cloned porcine leukocyte 12-LO in S. frugiperda cells and purification by Ni2+-nitrilotriacetate chromatography provides a straightforward method for isolation of milligram quantities of this form of 12-LO. Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Baculoviridae; Cells, Cultured; Chromatography, Affinity; Cloning, Molecular; Histidine; Hydroxyeicosatetraenoic Acids; Kinetics; Leukocytes; Moths; Nitrilotriacetic Acid; Organometallic Compounds; Peptides; Precipitin Tests; Protein Engineering; Recombinant Fusion Proteins; Substrate Specificity; Swine | 1994 |