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

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