Interaction of leukotriene C₄ and chinese hamster lung fibroblasts (V79A03 cells). 2. Subcellular distribution of binding and unlikely role of glutathione-S-transferase

It was reported previously that radiation-induced cytotoxicity in V79A03 (V79) cells was attenuated by pretreatment of cells with leukotriene C₄ (LTC₄), leading us to determine that V79 cells possessed specific binding sites, with characteristics of receptors, for LTC₄ (see the preceding, companion communication). Additional studies were conducted to determine the subcellular distribution and the chemical nature of the LTC₄ binding site in V79 cells. Trypsin treatment of cells before LTC, binding assays resulted in a 74% reduction in high-affinity binding. In tests to examine the subcellular location of LTC₄ binding, plasma membrane and nuclear fractions were obtained from V79 cells. In contrast to Scatchard analyses of LTC₄ binding to intact cells which were curvilinear, Scatchard analyses of nuclear and plasma membrane fractions were linear, indicative of the presence in these cellular substituents of low and high-affinity binding, respectively. To examine the nature of the high-affinity LTC₄ binding sites, intact V79 cells were photolyzed with [³H]-LTC₄ rendered photoactive by preincubation with N-hydroxysuccinimidyl-4-azidobenzoate. The cell-bound radioactivity migrated during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of approaximately 40 kdal. Five different commercial preparations of glutathione-S-transferase (GST), which has been implicated as a source of LTC₄ "specific binding" in other cells, migrated in the same SDS_PAGE system with an apparent molecular weigth of 20-24 kdal. Furthermore, preincubations of V79 cells with three antisera generated against GST had minimal effects upon subsequent LTC₄ binding to intact cells. These data, suggest that the radioprotective effect of LTC₄ upon V79 cells may be attributable to a receptor-mediated phenomenon which appears distinct from leukotriene binding to GST.