Abstract
Recently we demonstrated that Lewis lung (3LL) tumor cells express 12-lipoxygenase (12-LOX) mRNA and protein, respectively. In this study we partially sequenced the 12-LOX cDNA after reverse-transcription polymerase chain reaction amplification of 12-LOX mRNA from cultured 3LL cells. Comparison with platelet and leukocyte 12-LOX indicates that 3LL 12-LOX is identical with the platelet-type enzyme at least within the sequenced region. Further, we investigated the intracellular distribution of both 12-LOX enzyme protein and its activity which are prerequisites for understanding 12-LOX regulation. 12-LOX activity was monitored via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance liquid chromatography. 12-LOX protein was measured by direct slot blot and by Western Blotting. In 3LL cells, both 12-LOX activity and 12-LOX protein were predominantly localized in the cytosol. This 12-LOX activity was optimal at 37°C. However at 24°C and 10°C, it showed 87% and 61% of this activity, respectively, thus differing distinctly from 12-LOX in platelets or rat basophilic leukemia cells. Incubation of 3LL cell homogenates with 0–100 μM free Ca 2+ and subsequent separate analyses of cytosol and membrane fractions indicated that, as in platelets, an increase in intracellular free Ca 2+ caused a loss of cytosolic 12-LOX activity. However, no significant Ca 2+-induced increase in membrane-associated 12-LOX activity was observed under these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca 2+-dependent loss in the cytosol and a concomitant increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca 2+-dependent manner, but is no longer active or becomes inactivated at the membrane site.
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