Purification and characterization of active human interleukin-1 beta-converting enzyme from THP.1 monocytic cells.
Interleukin-1 beta-converting enzyme (ICE) was purified from dialyzed cytoplasmic extracts of THP.1 human monocytic cells by a combination of DEAE-5PW and SP-5PW ion exchange and C4 reverse phase high performance liquid chromatography. Sequence information from tryptic and Asp.N peptides on the isolated 20-kDa (p20) and a 10-kDa (p10) proteins enabled the subsequent cloning of ICE (Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774) and localized the active site Cys. Immunoblots with ICE specific antibodies and NH2-terminal sequencing indicated that ICE active column fractions contained in addition to p20 and p10 an alternatively processed form of the p20 protein (p22) containing an extra 16 amino acids NH2-terminal to the p20. Furthermore, immunoblot analysis of the ion exchange column effluent showed that p20 and p22 were found together in three separate fractions distinguished by differences in p10: an intact p10 with complete ICE activity, a COOH-terminally truncated form of p10 with decreased ICE activity, and an absence of p10 with no ICE activity. These results indicate that the p10 protein is essential for ICE activity and that the ICE holoenzyme contains an intact p10 subunit paired with a p20 or p22 catalytic subunit.
Miller
D K
DK
Department of Biochemical and Molecular Pathology, Merck Research Laboratories, Rahway, New Jersey 07065.
Ayala
J M
JM
Egger
L A
LA
Raju
S M
SM
Yamin
T T
TT
Ding
G J
GJ
Gaffney
E P
EP
Howard
A D
AD
Palyha
O C
OC
Rolando
A M
AM
D000595:Amino Acid Sequence; D000906:Antibodies; D020170:Caspase 1; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes; D010446:Peptide Fragments / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000906:Antibodies; D010446:Peptide Fragments; D008715:Methionine; D010450:Endopeptidases; D020170:Caspase 1; D008666:Metalloendopeptidases