12-Lipoxygenase from rat basophilic leukemia cells: separation from 5-lipoxygenase and temperature-dependent inactivation by hydroperoxy fatty acid.

12-Lipoxygenase and 5-lipoxygenase from rat basophilic leukemia cells were separated by protein-HPLC in a single step. Upon incubation in the presence of Ca2+, 12-lipoxygenase converted arachidonic acid into 12(S)-hydroxyeicosatetraenoic acid and linoleic acid into 13(S)-hydro(pero)xyoctadecadienoic acid. The reaction products were analyzed by reversed-phase and chiral straight-phase HPLC with ultraviolet-detection. Using the cytosolic fraction of rat basophilic leukemia cells, optimal 12-lipoxygenase activity was observed at 10 degrees C. At 37 degrees C 12-lipoxygenase was very rapidly inactivated by its own product, hydroperoxy fatty acid, at low concentrations (10-100 nM).