[34] Leukocyte arachidonate 5-lipoxygenase: Isolation and characterization

Publisher Summary The chapter presents a study on isolation and characterization of leukocyte arachidonate 5-lipoxygenase. The chapter discusses the method of assay stating that there are a number of issues that complicate the assay of leukocyte arachidonate 5-1ipoxygenase. The purification procedure described in the chapter was developed for the 5- lipoxygenase from human peripheral blood leukocytes. Unlike the 5-1ipoxygenase of rat and pig, purification of the human leukocyte 5-1ipoxygenase has revealed a number of cellular fractions that stimulate 5-1ipoxygenase product formation in the routine high-performance liquid chromatography (HPLC) assay. In regard to the properties of 5-lipoxygenase, because of limited availability and poor stability, very few detailed kinetic studies have been performed with purified leukocyte 5-1ipoxygenase. Investigations performed with partially purified samples suggest a deviation from Michaelis-Menten behavior, with substrate inhibition observed. As has been shown for the lipoxygenase from soybeans, leukocyte 5- lipoxygenase displays a kinetic lag phase that is eliminated by fatty acyl hydroperoxides, and exacerbated by reducing agents, such as sulfhydryl compounds. This suggests that, like the soybean enzyme, leukocyte 5-1ipoxygenase may undergo a product-mediated oxidative activation step; however, the chemical mechanism of such a step remains to be elucidated.