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).
van der Donk
E M
EM
Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Verhagen
J
J
Veldink
G A
GA
Vliegenthart
J F
JF
D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000302:isolation & purification / Q000378:metabolism*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D005227:Fatty Acids / Q000378:metabolism; D007942:Leukemia, Experimental / Q000201:enzymology*; D051381:Rats; D013696:Temperature; D014407:Tumor Cells, Cultured
D001095:Arachidonic Acids; D005227:Fatty Acids; D016718:Arachidonic Acid; D001092:Arachidonate 12-Lipoxygenase