ACTIVITY AND PROTEIN DISTRIBUTION OF 12-LIPOXYGENASE IN HEL CELLS - INDUCTION OF MEMBRANE-ASSOCIATION BY PHORBOL ESTER TPA, MODULATION OF ACTIVITY BY GLUTATHIONE AND 13-HPODE, AND CA2-DEPENDENT TRANSLOCATION TO MEMBRANES()

The understanding of the intracellular regulation of 12-lipoxygenase r equires a knowledge of the distribution of both enzyme protein and its activity. In human erythroleukemia cells, the membrane fraction conta ins about 90% of the total cellular 12-lipoxygenase activity, whereas only approximately 10% of 12-lipoxygenase activity resides in the cyto sol. However, the majority of the cellular 12-lipoxygenase protein is found in the cytosol. Pretreatment of cells for 0-3 days with 160 nM T PA caused a marked, time-dependent increase in membrane-bound 12-lipox ygenase activity and protein, respectively. In contrast, the cytosolic amount of 12-lipoxygenase protein and activity, respectively, were mi nimally altered by this TPA treatment. Recombining the active membrane fraction with cytosol resulted in no significant inhibition of its 12 -lipoxygenase activity, but the addition of GSH to the membrane fracti on inhibited 12-lipoxygenase activity in a dose-dependent manner. On t he other hand, the cytosolic enzyme can be rendered active in the pres ence of 1 mu M 13-hydroperoxyoctadecadienoic acid. in HEL cell homogen ates, a partial translocation of the cytosolic enzyme to the membrane takes place in a Ca2+-dependent manner, resulting in an increase in me mbrane-associated 12-lipoxygenase activity and a concomitant decrease in cytosolic 12-lipoxygenase activity above 0.1 mu M Ca2+.