12-LIPOXYGENASE IN LEWIS LUNG-CARCINOMA CELLS - MOLECULAR IDENTITY, INTRACELLULAR-DISTRIBUTION OF ACTIVITY AND PROTEIN, AND CA2-DEPENDENT TRANSLOCATION FROM CYTOSOL TO MEMBRANES()

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 polym erase chain reaction amplification of 12-LOX mRNA from cultured 3LL ce lls. 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 distribut ion of both 12-LOX enzyme protein and its activity which are prerequis ites for understanding 12-LOX regulation. 12-LOX activity was monitore d via the production of 12-hyroxyeicosatetraenoic acid from 3LL cells and their subcellular fractions using reverse-phase high performance l iquid 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 acti vity was optimal at 37 degrees C. However at 24 degrees C and 10 degre es C, it showed 87% and 61% of this activity, H respectively thus diff ering distinctly from 12-LOX in platelets or rat basophilic leukemia c ells. Incubation of 3LL cell homogenates with 0-100 mu M free Ca2+ and subsequent separate analyses of cytosol and membrane fractions indica ted that, as in platelets, an increase in intracellular flee Ca2+ caus ed a loss of cytosolic 12-LOX activity. However, no significant Ca2+-i nduced increase in membrane-associated 12-LOX activity was observed un der these conditions in 3LL cells. In contrast, at the 12-LOX protein level we observed a Ca2+-dependent loss in the cytosol and a concomita nt increase in the membrane fraction. Thus, we suggest that 12-LOX in 3LL cells undergoes rapid translocation from cytosol to membrane in a Ca2+-dependent manner, but is no longer active or becomes inactivated at the membrane site.