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The Rat Leukocyte-type 12-Lipoxygenase Exhibits an Intrinsic Hepoxilin A3 Synthase Activity

Nigam, Santosh ; Patabhiraman, Shankarnarayanan ; Ciccoli, Roberto ; Ishdorj, Ganchimeg ; Schwarz, Kristin ; Petrucev, Branka ; Kühn, Hartmut ; Haeggström, Jesper Z.

The Journal of biological chemistry, 2004-07, Vol.279 (28), p.29023-29030 [Periódico revisado por pares]

United States: American Society for Biochemistry and Molecular Biology

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Título:
The Rat Leukocyte-type 12-Lipoxygenase Exhibits an Intrinsic Hepoxilin A3 Synthase Activity
Autor: Nigam, Santosh ; Patabhiraman, Shankarnarayanan ; Ciccoli, Roberto ; Ishdorj, Ganchimeg ; Schwarz, Kristin ; Petrucev, Branka ; Kühn, Hartmut ; Haeggström, Jesper Z.
Assuntos: Amino Acid Sequence ; Animals ; Arachidonate 12-Lipoxygenase - genetics ; Arachidonate 12-Lipoxygenase - metabolism ; Arachidonate 15-Lipoxygenase - metabolism ; Arachidonate 5-Lipoxygenase - metabolism ; Arachidonic Acids - chemistry ; Arachidonic Acids - metabolism ; Cell Line, Tumor ; Humans ; Insulinoma - metabolism ; Insulinoma - pathology ; Intramolecular Oxidoreductases - genetics ; Intramolecular Oxidoreductases - metabolism ; Isoenzymes - genetics ; Isoenzymes - metabolism ; Leukocytes - enzymology ; Medicin och hälsovetenskap ; Molecular Conformation ; Molecular Sequence Data ; Molecular Structure ; Mutagenesis, Site-Directed ; Rabbits ; Rats ; Sequence Alignment
É parte de: The Journal of biological chemistry, 2004-07, Vol.279 (28), p.29023-29030
Descrição: Hepoxilins are biologically relevant eicosanoids formed via the 12-lipoxygenase pathway of the arachidonic acid cascade. Although these eicosanoids exhibit a myriad of biological activities, their biosynthetic mechanism has not been investigated in detail. We examined the arachidonic acid metabolism of RINm5F rat insulinoma cells and found that they constitutively express a leukocyte-type 12 S -lipoxygenase. Moreover, we observed that RINm5F cells exhibit an active hepoxilin A 3 synthase that converts exogenous 12 S -HpETE (12 S -5Z,8-Z,10 E ,14Z-12-hydro(pero)xy-eicosa-5,8,10,14-tetraenoic acid) or arachidonic acid predominantly to hepoxilin A 3 . 12 S -lipoxygenase and hepoxilin A 3 synthase activities were co-localized in the cytosol; immunoprecipitation with an anti-12 S -lipoxygenase antibody co-precipitated the two catalytic activities. These data suggested that hepoxilin A 3 synthase activity may be considered an intrinsic catalytic property of the leukocyte-type 12 S -lipoxygenase. To test this hypothesis we cloned the leukocyte-type 12 S -LOX from RINm5F cells, expressed it in Pichia pastoris , and found that the recombinant enzyme exhibited both 12 S -lipoxygenase and hepoxilin A 3 synthase activities. The recombinant human platelet-type 12 S -lipoxygenase and the porcine leukocyte-type 12 S -lipoxygenase also exhibited hepoxilin A 3 synthase activity. In contrast, the native rabbit reticulocyte-type 15 S -lipoxygenase did not convert 12 S -HpETE to hepoxilin isomers. These data suggest that the positional specificity of lipoxygenases may be crucial for this catalytic function. This hypothesis was confirmed by site-directed mutagenesis studies that altered the positional specificity of the rat leukocyte-type 12 S - and the rabbit reticulocyte-type 15-lipoxygenase. In summary, it may be concluded that naturally occurring 12 S -lipoxygenases exhibit an intrinsic hepoxilin A 3 synthase activity that is minimal in lipoxygenase isoforms with different positional specificity.
Editor: United States: American Society for Biochemistry and Molecular Biology
Idioma: Inglês

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The Rat Leukocyte-type 12-Lipoxygenase Exhibits an Intrinsic Hepoxilin A3 Synthase Activity

Nigam, Santosh ; Patabhiraman, Shankarnarayanan ; Ciccoli, Roberto ; Ishdorj, Ganchimeg ; Schwarz, Kristin ; Petrucev, Branka ; Kühn, Hartmut ; Haeggström, Jesper Z.

United States: American Society for Biochemistry and Molecular Biology

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