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Analysis of the human and mouse genome sequences has enabled a detailed analysis of the structure and organization of the lipoxygenase genes in the respective species. Humans appear to possess six functional genes and at least three pseudogenes while mice have seven functional genes. The arrangement of the genes is quite similar between the species with most of the human lipoxygenase genes appearing on the short arm of chromosome 17 and in mice on the syntenic portion of chromosome 11. The 5-lipoxygenase gene is unique in several respects including its distinct separate chromosomal localization and its size (4–7× larger than other lipoxygenase genes). Three of the seven murine lipoxygenase genes have been disrupted by gene targeting. While the knockout mice appear outwardly normal, a number of important findings have been discovered using these mice and these will be covered in this review. [Copyright &y& Elsevier]

A baculoviral expression vector consisting of a sequence encoding a six-histidine tag apposed to the human platelet 12-lipoxygenase cDNA, under control of the polyhedrin promoter, was constructed. Recombinant 12-lipoxygenase baculoviruses were used to infect Spodoptera frugiperda insect cells (Sf9). At 54 h post-infection, maximal 12-lipoxygenase activity and protein levels were achieved: the enzyme was purified to apparent homogeneity in a single step by nickel-ion-chelation chromatography in which the (His)6-tagged 12-lipoxygenase was eluted with 100 mM imidazole. The purified enzyme metabolized arachidonic acid almost exclusively to 12-hydroperoxyeicosatetracnoic acid with little, if any. epoxyalcohol or reduction products and had a Vmax of 2–4 μmol min-1mg protein-1, Km, of 10 μM and keat of ≈250 min-1. Linoleic acid, on the other hand, was converted to (13S)-13-hydroperoxy-octadecadienoic acid at a rate which was about 2% of that obtained with arachidonic acid as substrate, but displayed the same Km. The enzyme was most active between pH 7.5–8 and activity was stimulated significantly in the presence of 0.006% Tween-20. A polyclonal antibody to the recombinant enzyme was generated and found to recognize a single 75-kDa band in platelets, human erythroleukemia cells and 12-1ipoxygenase baculoviral-infected Sf9 cells by immunoblot and immunoprecipitation methods. 12-Lipoxygenase protein represented 0.1% of the total soluble protein in platelet preparations. In immunofluorescence experiments 12-lipoxygenase was observed in the cytoplasm of infected insect cells and in the human megakaryoblastic DAMI cell line. The isolation of large quantities of pure human platelet 12-1ipoxygenase should facilitate detailed biochemical structure/function studies. [ABSTRACT FROM AUTHOR]