The 1.4-MDa apoptosome is a critical intermediate in apoptosome maturation.
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 approximately 700-kDa apoptosome matures through a 1.4-MDa intermediate before a approximately 700-kDa apoptosome is reformed and procaspase-3 is activated. We further demonstrate that 150 mM KCl interferes with the conversion of the initial approximately 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.
Beem
Elaine
E
Division of Nephrology, Hypertension and Transplantation, Department of Medicine, University of Florida, PO Box 100224, Gainesville, FL 32610, USA.
Holliday
L Shannon
LS
Segal
Mark S
MS
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053477:Apoptotic Protease-Activating Factor 1; D053453:Caspase 9; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D045304:Cytochromes c / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D019169:Jurkat Cells; D008954:Models, Biological; D011189:Potassium Chloride / Q000494:pharmacology; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D011506:Proteins; D011994:Recombinant Proteins; D011189:Potassium Chloride; D045304:Cytochromes c; C506433:CASP9 protein, human; D053453:Caspase 9; D020169:Caspases