Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation

doi:10.1084/jem.180.6.2113

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Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation

SC Wright, QS Wei, J Zhong, H Zheng, DH Kinder and JW Larrick
Palo Alto Institute of Molecular Medicine, Mountain View, California 94043.

We report the purification of a protease from tumor cells undergoing apoptosis that is involved in activating DNA fragmentation. Initial studies revealed that two inhibitors of serine proteases, N-1- tosylamide-2-phenylethylchloromethyl ketone and carbobenzoxy-Ala-Ala- borophe (DK120), suppressed tumor necrosis factor or ultraviolet (UV) light-induced DNA fragmentation in the U937 histiocytic lymphoma as well as UV light-induced DNA fragmentation in the BT-20 breast carcinoma, HL-60 myelocytic leukemia, and 3T3 fibroblasts. The protease was purified by affinity chromatography with DK120 as ligand and showed high activity on a synthetic substrate preferred by elastase-like enzymes (Ala-Ala-Pro-Val p-nitroanilide), but was inactive on the trypsin substrate, N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester, or the chymotrypsin substrate, Ala-Ala-Pro-Phe p-nitroanilide. The activity of the DK120-binding protease purified from U937 cells undergoing apoptosis was increased approximately 10-fold over that recovered from normal cells. Further purification to homogeneity by heparin-Sepharose affinity chromatography followed by reverse phase high-performance liquid chromatography revealed a single band of 24 kD on a silver-stained sodium dodecyl sulfate gel. In addition to protease activity, the purified enzyme induced DNA fragmentation into multiples of 180 basepairs in isolated U937 nuclei. These findings suggest the 24- kD protease is a novel enzyme that activates DNA fragmentation in U937 cells undergoing apoptosis.
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