FIGURE 4.

Time dependence of arachidonic acid oxygenation by purified wild-type human ALOX15 and its T560M mutant. Wild-type and T560M mutant were expressed and purified as described under “Materials and Methods.” LOX concentrations of each preparation were quantified by immunoblotting, and in this experiment, the amount of T560M used was 5-fold higher than the wild type. Activity assays were carried out as described, and the reaction was stopped by the addition of 500 μl of ice-cold methanol. After reduction of hydroperoxides and acidification, the samples were centrifuged, and aliquots of the supernatant were used for HPLC analysis.