The functional and biochemical properties of cytoplasmic granules of rat large granular lymphocyte tumors have been further delineated. Antibodies against the two BLT esterases purified to homogeneity were used to show that these proteins are serologically not cross-reactive, confirming previous enzymatic inhibition data. The possible interactions of these enzymes in cytolysin function was examined by mixing the pure LGL granule cytolysin and the pure major BLT esterase and testing for lytic activity. On tumor target cells (but not red cells), a synergistic effect was observed which could increase the cytolysin potency as much as ten-fold. Target preincubation with the enzyme did not cause this effect, nor was it seen with the minor BLT esterase. While these results suggest that the major enzyme can potentially increase the lytic efficiency of cytotoxic lymphocytes, the mechanism of this effect is still unclear. The ability of granules of cytotoxic lymphocytes to release DNA from target nuclei was studied in order to explain the rapid DNA degradation seen when cytotoxic lymphocytes attack target cells. Purified LGL tumor granules cause nuclear DNA release along with lysis of tumor cells, while the purified cytolysin causes lysis with no DNA breakdown. Two additional granule components cause DNA release from detergent permeabilized cells, but these appear to be minor granule proteins. Our results suggest that target cell DNA breakdown can be accounted for by the granule exocytosis mechanism. In order to begin a molecular biology approach to LGL granule proteins, a cDNA library in lambda gt10 was constructed from rat LGL tumor cell mRNA. The purified cytolysin protein was subjected to cyanogen bromide digestion and the resulting peptides separated by HPLC. Sequences of 25 amino acid residues of two of these were obtained by Edman degradation. Based on these sequences, synthetic oligonucleotide probes were synthesized in order to probe the cDNA library for the cytolysin clone.
