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J Biol Chem, Vol. 275, Issue 9, 6067-6070, March 3, 2000

ACCELERATED PUBLICATION
Apaf-1 Oligomerizes into Biologically Active ~700-kDa and Inactive ~1.4-MDa Apoptosome Complexes^*

Kelvin Cain, Shawn B. Bratton, Claudia Langlais, Gail Walker, David G. Brown, Xiao-Ming Sun, and Gerald M. Cohen^§

From the MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester, LE1 9HN United Kingdom

Apaf-1, by binding to and activating caspase-9, plays a critical role in apoptosis. Oligomerization of Apaf-1, in the presence of dATP and cytochrome c, is required for the activation of caspase-9 and produces a caspase activating apoptosome complex. Reconstitution studies with recombinant proteins have indicated that the size of this complex is very large in the order of ~1.4 MDa. We now demonstrate that dATP activation of cell lysates results in the formation of two large Apaf-1-containing apoptosome complexes with M_r values of ~1.4 MDa and ~700 kDa. Kinetic analysis demonstrates that in vitro the ~700-kDa complex is produced more rapidly than the ~1.4 MDa complex and exhibits a much greater ability to activate effector caspases. Significantly, in human tumor monocytic cells undergoing apoptosis after treatment with either etoposide or N-tosyl-l-phenylalanyl chloromethyl ketone (TPCK), the ~700-kDa Apaf-1 containing apoptosome complex was predominately formed. This complex processed effector caspases. Thus, the ~700-kDa complex appears to be the correctly formed and biologically active apoptosome complex, which is assembled during apoptosis.

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^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a Glaxo Wellcome Post Graduate studentship award.

^§ To whom correspondence should be addressed: MRC Toxicology Unit, Hodgkin Bldg., University of Leicester, P. O. Box 138, Lancaster Rd., Leicester, LE1 9HN UK. Tel.: 0116 252 5601; Fax: 0116 252 5616; E-mail: gmc2@le.ac.uk.

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Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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