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Publication : cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases.

First Author  Chen XS Year  1994
Journal  J Biol Chem Volume  269
Issue  19 Pages  13979-87
PubMed ID  8188678 Mgi Jnum  J:18190
Mgi Id  MGI:66203 Doi  10.1016/s0021-9258(17)36743-1
Citation  Chen XS, et al. (1994) cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases. J Biol Chem 269(19):13979-87
abstractText  Two types of 12-lipoxygenase that catalyze the transformation of arachidonic acid to 12(S)-hydroperoxyeicosatetraenoic acid (12-HPETE) have been previously classified into platelet-type and leukocyte-type categories. Here, we document, for the first time, a molecular characterization of both forms within the same species. The amino acid sequence of the murine platelet 12-lipoxygenase deduced from its cDNA is 58% identical to the murine spleen/leukocyte 12-lipoxygenase. Expression constructs carrying the cDNAs for the two 12-lipoxygenase forms were introduced into human embryonic kidney 293 cells. The platelet-type enzyme metabolized arachidonic acid exclusively to 12-HPETE, whereas the leukocyte-type enzyme formed both 12-HPETE and 15-hydro(pero)xyeicosatetraenoic acid in a ratio of approximately 3:1. Linoleic acid was metabolized to a similar extent by the latter enzyme to 13-hydro(pero)xyoctadecadienoic acid but not by the platelet enzyme. Mutagenesis and deletion of the highly conserved lipoxygenase C-terminal isoleucine (Ile663), a residue believed to be involved in the non-heme iron atom coordination of all lipoxygenases, was performed. Deletion of Ile663 and substitution with most amino acids abolished enzyme activity. Only a valine substitution retained significant activity. These findings would tend to indicate a stringent requirement for the proper spatial alignment and folding of the C-terminal chain back into the core of the enzyme to interact with the iron atom by analogy with the recently determined crystal structure of a soybean lipoxygenase (Boyington, J. C., Gaffney, B. J., and Amzel, L. M. (1993) Science 260, 1482-1486). The platelet-type and leukocyte-type 12-lipoxygenase genes were cloned from a murine 129 Sv genomic library. Both genes are divided into a similar 14-exon/13-intron format, with the platelet-type gene being approximately twice the size of the leukocyte-type gene (13 versus 7.5 kilobases). A segment of a third gene was also isolated and probably represents a pseudogene derivative of either of these 12-lipoxygenase genes. All three genes were mapped to the central region of mouse chromosome 11 in a region of homology with human chromosome 17. Antibodies prepared against the two forms of 12-lipoxygenase revealed the differential distribution of the two enzymes throughout the mouse.
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