Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation.
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.
Palo Alto Institute of Molecular Medicine, Mountain View, California 94043.
S C
SC
Wright
Q S
QS
Wei
J
J
Zhong
H
H
Zheng
D H
DH
Kinder
J W
JW
Larrick
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001943:Breast Neoplasms; D002460:Cell Line; D002846:Chromatography, Affinity; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D016403:Lymphoma, Large B-Cell, Diffuse; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D003851:Deoxyribonucleases; D010450:Endopeptidases