The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation

1 Division of Nephrology, Hypertension and Transplantation, Department of Medicine; and 2 College of Dentistry, University of Florida, Gainesville, Florida 32610 Submitted 3 June 2003 ; accepted in final form 27 April 2004 Previously, we demonstrated that both 150 mM KCl and alkaline pH inhibit cytochrome c -mediated activation of procaspase-3 in a unique manner. To determine the mechanism of inhibition, we analyzed the effect of KCl and alkaline pH on the formation of apoptosomes (a large complex consisting of cytochrome c , Apaf-1, and procaspase-9/caspase-9) in vitro. Our results suggest that an initial 700-kDa apoptosome matures through a 1.4-MDa intermediate before a 700-kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrate that 150 mM KCl interferes with the conversion of the initial 700-kDa apoptosome to the 1.4-MDa intermediate, while alkaline pH "traps" the apoptosome in the 1.4-MDa intermediate. Analysis of the cleaved state of procaspase-9 and procaspase-3 suggests that the 1.4-MDa intermediate may be required for cleavage of procaspase-9. Consistent with these results, in vivo data suggest that blocking acidification during the induction of apoptosis inhibits activation of procaspase-3. On the basis of these results, we propose a model of apoptosome maturation. caspase; pH; potassium; apoptosis Address for reprint requests and other correspondence: M. S. Segal, PO Box 100224, Gainesville, FL 32610 (E-mail: segalms{at}medicine.ufl.edu ).