Necrosin: purification and properties of a cytotoxin derived from a murine macrophage-like cell line.

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An acidic proteinaceous cytotoxin was isolated from the serum-free, cell-free supernatants of J774.1 (a murine macrophage-like line) cells that had been treated with bacterial endotoxin. Cytotoxic activity was routinely monitored using the sensitive murine tumorigenic fibroblast line L-M. The toxin was purified 8000-fold by ion-exchange and electrophoretic procedures. It was purified to greater than 90% homogeneity, as assessed by photometric scanning of silver-stained NaDodSO4/polyacrylamide gels. The specific activity of the purified toxin was 32,000 units/microgram. The toxin was composed of self-aggregating non-sulfhydryl-linked multimers of a Mr 15,000 subunit. The pI of the monomer was 4.6. The active multimeric forms, as assessed by gel filtration and assayed using L-M cells, were of Mr 70,000 and 55,000. These forms were identical to those observed both in crude supernatants and in purified fractions that had not been subjected to denaturing agents. We call these forms "holotoxins" and conclude that they are aggregates of the Mr 15,000 protein. The purified toxin (1-1000 pM, 0.06-60 ng/ml) was active against a random assortment of tumorigenic and normal cell lines of murine, bovine, and human origin. For example, the diploid bovine endothelial line CPAE was nearly as sensitive as the L-M line. Similarities to other toxic macrophage products, lymphotoxin, and tumor necrosis factor are discussed.