CDNA CLONING, EXPRESSION, MUTAGENESIS, INTRACELLULAR-LOCALIZATION, AND GENE CHROMOSOMAL ASSIGNMENT OF MOUSE 5-LIPOXYGENASE

5-Lipoxygenase of mouse macrophages and bone marrow derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined wit h confocal microscopy provided evidence for distinct intracellular exp ression patterns and trafficking of 5-lipoxygenase upon cellular activ ation. In resting BMMC, 5-lipoxygenase was found within the nucleus co -localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antige n-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the in cubation medium, or calcium ionophore A23187 challenge, altered the pr edominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was clone d by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mi ce it was determined that a single isoform accounts for both soluble a nd membrane bound and nuclear and cytosolic-localized enzyme in macrop hages and BMMC. A mutation at amino acid 672 (Val --> Met) introduced serendipitously during the cloning process was found to completely abo lish 5-lipoxygenase enzyme activity when the enzyme was expressed in h uman embryonic kidney 293 cells. This subtle change is proposed to aff ect the ability of the COOH-terminal isoleucine to coordinate the esse ntial non-heme iron atom. In macrophages and BMMC obtained from 5-lipo xygenase-deficient mice, compensatory changes in expression of genes i nvolved in the biosynthesis of leukotriene B-4 were investigated. 5-Li poxygenase-activating protein expression was reduced by 50%, while leu kotriene A(4) hydrolase expression was unaltered. The 5-lipoxygenase g ene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcr oss analysis. These studies provide a global picture of the murine 5-l ipoxygenase system and raise questions about the role of 5-lipoxygenas e and leukotrienes within the nucleus.