Cytochrome c Promotes Caspase-9 Activation by Inducing Nucleotide Binding to Apaf-1

Abstract

We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1·cytochromec complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochromec-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (β,γ-methylene adenosine 5′-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1. We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1·cytochromec complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochromec-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (β,γ-methylene adenosine 5′-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1. apoptotic protease-activating factor-1 adenosine 5′-γ-thiotriphosphate β,γ-methylene adenosine 5′-triphosphate bovine serum albumin polyacrylamide gel electrophoresis thin layer chromatography Caspases are a group of intracellular cysteine proteases that cleave protein substrates after aspartic acid residues, generating the characteristic morphological changes associated with apoptosis (1Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). Because caspase-mediated proteolysis is in general an irreversible process, caspases are synthesized as inactive pro-forms that are activated only during apoptosis (1Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar). One major intracellular caspase activation pathway is triggered by cytochrome c released from mitochondria (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). Once in cytosol, cytochrome c binds its cytosolic partner Apaf-1,1 the human homologue of Caenorhabditis elegans apoptotic protein CED-4, and induces the oligomerization of Apaf-1·cytochrome c complex in a dATP/ATP-dependent manner (3Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 4Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar, 6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (410) Google Scholar, 8Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). This multimeric complex, namely apoptosome, is sufficient to recruit the initiator caspase, caspase-9, to the complex and induces procaspase-9 autoactivation (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar). The activated caspase-9 then cleaves and activates other downstream caspases such as caspase-3 and caspase-7 that constitute major caspase activity in apoptotic cells (3Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 4Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar, 6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (410) Google Scholar, 8Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). Although this mitochondria-initiated caspase activation pathway is supported by many biochemical and genetic experiments (9Cecconi F. Alvarez-Bolado G. Meyer B.I. Roth K.A. Gruss P. Cell. 1998; 94: 727-737Abstract Full Text Full Text PDF PubMed Scopus (823) Google Scholar, 10Yoshida H. Kong Y.Y. Yoshida R. Elia A.J. Hakem A. Hakem R. Penninger J.M. Mak T.W. Cell. 1998; 94: 739-750Abstract Full Text Full Text PDF PubMed Scopus (1004) Google Scholar, 11Kuida K. Haydar T.F. Kuan C.Y. Gu Y. Taya C. Karasuyama H. Su M.S. Rakic P. Flavell R.A. Cell. 1998; 94: 325-337Abstract Full Text Full Text PDF PubMed Scopus (1460) Google Scholar, 12Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A. de la Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe S.W. Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1171) Google Scholar), the detailed molecular mechanism remains elusive. For example, it is well established that dATP or ATP is required for the apoptosome formation (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 3Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 4Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar, 5Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Alnemri E.S. Mol. Cell. 1998; 1: 949-957Abstract Full Text Full Text PDF PubMed Scopus (969) Google Scholar, 6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (410) Google Scholar, 8Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). It is not clear, however, whether the hydrolysis or simply binding of nucleotides to Apaf-1 is critical for such an event. Additionally, the relationship between nucleotide and cytochromec binding to Apaf-1 is still not established, although both are absolutely needed for the reaction (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). In the current manuscript, we report the study of molecular mechanism of Apaf-1-mediated caspase-9 activation using highly purified recombinant Apaf-1 and procaspase-9. The results indicate that the key event in this caspase activation reaction is nucleotide binding to Apaf-1. The event is regulated by cytochrome c as well as procaspase-9. Nucleotides dATP, ATP, dGTP, and ATPγS were purchased from Amersham Pharmacia Biotech; dADP and ADPCP were from Sigma. [α-33P]dATP was obtained from NEN Life Science Products, and [γ-32P]dATP from ICN. Polyclonal antibodies against Apaf-1 and caspase-9 were prepared as described previously (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar). A monoclonal antibody against cytochrome cwas purchased from PharMingen. Purified horse cytochromec was produced as described previously (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar).35S-labeled procaspase-3 was in vitro translated and purified as described previously (2Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar). Recombinant procaspase-9, both wild type and D315A mutant, was expressed and purified as described previously (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar). Recombinant Apaf-1 was expressed and nickel affinity-purified as described previously (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar), followed by MonoQ chromatography using a fast protein liquid chromatography system (FPLC, Amersham Pharmacia Biotech). The MonoQ column was equilibrated with buffer A (20 mm Hepes-KOH, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride) containing 100 mm NaCl, and subsequently eluted with a 20-ml linear gradient from 100 mm NaCl to 250 mm NaCl in buffer A. Apaf-1 was eluted at ∼200 mm NaCl. After being dialyzed against buffer A, the purified Apaf-1 was stored in aliquots at −80 °C. A rapid filter binding assay was developed to measure dATP binding to Apaf-1. In a final volume of 20 μl in buffer A, containing additionally 1 mm MgCl2 and 1 mg/ml BSA, 500 nm [α-33P]dATP (100–200 cpm/fmol) was mixed with other factors as indicated in the figure legends. The reaction was started by adding 100 nm Apaf-1 and incubated at 30 °C for 15 min. After incubation, the samples were diluted immediately with 2 ml of chilled washing buffer (20 mmTris-Cl, pH 8.0, 100 mm NaCl, and 40 mmMgCl2) and filtered through 25-mm BA85 nitrocellulose filters (Schleicher and Schuell). Filters were washed twice with 2 ml of chilled washing buffer and counted for radioactivity with a scintillation counter (Beckman). For K D measurement, the same procedure was performed except that: 1) different amounts of dATP were used in each reaction as indicated in the presence of 500 nm cytochrome cor 500 nm cytochrome c plus 50 nmD315A procaspase-9; and 2) the mixtures were incubated for 1 h, at which time point the binding equilibrium was reached.K D values were calculated by double-reciprocal plot (1/[dATP]total versus1/[Apaf-1·dATP]) analysis. Linear regression of the plots was processed automatically using SigmaPlot program. Radioactive samples were loaded on a Baker-flex cellulose polyethyleneimine TLC plate (J. T. Baker, Inc.) as indicated. One μl of 10 mm nonradioactive dATP and dADP were loaded as the controls. After being developed in 1 m formic acid plus 0.5m LiCl, the plate was air-dried. The control dATP and dADP were visualized under UV light in the dark, and the resolved radioactive samples were detected by phosphorimaging. In buffer A containing additionally 1 mm MgCl2 and 1 mg/ml BSA, 1 μm [γ-32P]dATP (50–100 cpm/fmol) was mixed with other factors as indicated in figures. The reaction was started by finally adding 100 nm Apaf-1 and was incubated at 30 °C. Hydrolysis of dATP was measured as the release of γ-32Pi from dATP, as described previously for the ATPase assay (13Mattsson J.P. Schlesinger P.H. Keeling D.J. Teitelbaum S.L. Stone D.K. Xie X.S. J. Biol. Chem. 1994; 269: 24979-24982Abstract Full Text PDF PubMed Google Scholar), with minor modifications. Briefly, at indicated times, 20-μl aliquots of samples were taken out from reactions and added to glass tubes containing 0.5 ml of 1.25n perchloric acid to terminate nucleotide hydrolysis, and then 0.125 ml of 5% (w/v) ammonium molybdate was added followed by the addition of 0.75 ml of 1:1 isobutanol benzene. The mixtures were vibrated by vortex to achieve phase extraction. After phase separation, 0.25-ml aliquots of the organic phase were counted for radioactivity by a scintillation counter, and dATP hydrolysis by Apaf-1 was calculated as the release of Pi from dATP. in our previously reported reconstitution of caspase-9 activation using purified recombinant Apaf-1 and procaspase-9 (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar), the purity was measured by Coomassie Blue staining, and minor contamination remained that precluded us from quantitatively measuring the nucleotide binding and hydrolysis by Apaf-1. To improve the assay conditions, Apaf-1 was subjected to an additional purification step so that it was pure by silver staining standard (Fig.1 A). When purified, procaspase-9 and cytochrome c were added to Apaf-1, and caspase-9 activation was observed in a time-dependent fashion measured by silver staining of the autocleaved 35-kDa subunit generated from the 50-kDa procaspase-9 (Fig. 1 A). Procaspase-9 activation requires the pre-formation of a multimeric Apaf-1·cytochrome c complex, apoptosome. To confirm the formation of apoptosome, the caspase-9 activation reaction mixture was separated in a gel filtration column, and the fractions from the column were analyzed by Western blot. The inactive Apaf-1 monomer ran at fraction 14 (Fig. 1 B; Ref. 6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar). However, as shown in Fig. 1 B, most of the Apaf-1 protein was migrated at a position correlating with a large complex (fractions 10 and 11). Cytochrome c and caspase-9 were also detected in these fractions. Interestingly, the majority of cleaved caspase-9 was not associated with the complex and was present in a free form that migrated at fractions 14–17. When caspase-9 activity was measured directly by adding 35S-labeled substrate procaspase-3, only the fractions that correlated with the large complex showed caspase-3 cleavage activity, whereas the major free caspase-9 fraction showed only marginal activity (Fig. 1 B, lower panel). This result further supports the model proposed by Lazebnik and colleague (14Rodriguez J. Lazebnik Y. Genes Dev. 1999; 13: 3179-3184Crossref PubMed Scopus (461) Google Scholar) that it is the holoenzyme consisting of Apaf-1 and caspase-9 that cleaves the downstream procaspases. the purified Apaf-1 and procaspase-9 allowed us to do a direct nucleotide binding assay using radiolabeled dATP. Nucleotide binding to proteins was measured by a filter binding assay. Procaspase-9 and cytochrome c showed no detectable binding of nucleotides by themselves (data not shown). Apaf-1 alone also showed little nucleotide binding activity, even though within the CED-4 homologous region Apaf-1 has the well conserved nucleotide binding site, Walker's A and B motif (Fig.2 B). However, when cytochromec was co-incubated with Apaf-1, nucleotide binding to Apaf-1 was significantly increased (Fig. 2, A and B). The nucleotide bound to Apaf-1 reached a plateau when cytochromec was used at 500 nm and higher concentrations (Fig. 2 A). as shown in Fig. 2 B, when procaspase-9 was dATP binding to Apaf-1 was stimulated Procaspase-9 cytochrome c no on dATP binding to Apaf-1. The of procaspase-9 not to to caspase-9 a cleavage procaspase-9 dATP binding as well as the wild type The of procaspase-9 on dATP binding to Apaf-1 reached a plateau at 50 nm procaspase-9 in the presence of cytochrome c (Fig. 2 of cytochrome procaspase-9 on dATP binding to Apaf-1. Nucleotide binding of Apaf-1 was measured as described under A, cytochrome c dATP binding to Apaf-1. of dATP to Apaf-1 was measured in the presence of different amounts of cytochrome c as procaspase-9 further dATP binding to Apaf-1 in a cytochrome manner. of dATP to Apaf-1 was measured in the presence of 100 nm cytochromec 50 nm procaspase-9, or the D315A as indicated. in the presence of 500 nm cytochrome c the of different amounts of D315A procaspase-9 on dATP binding to Apaf-1 was The binding of dATP to Apaf-1 in the presence of cytochromec or cytochrome c plus procaspase-9 were measured and calculated by double-reciprocal plot A). The K dATP binding to Apaf-1 in the presence of 500 c is The presence of procaspase-9 further the K to of the with cytochromec We also used this dATP binding assay to measure the binding of different shown in Fig. B, amounts of dATP with the radiolabeled dATP bound to Apaf-1. In and dADP not for dATP binding to Apaf-1 at ATP and ATPγS were and in for dATP binding to Apaf-1. To whether dATP hydrolysis is required for apoptosome formation and caspase activation after it binds to Apaf-1, we used [α-33P]dATP in the caspase-9 activation the reaction mixture on a gel filtration shown in Fig. A, after [α-33P]dATP with Apaf-1, cytochrome c, and procaspase-9, most of the Apaf-1 was in the apoptosome that also cytochrome c. The radioactive nucleotide showed when eluted from the One was at fractions 10 and correlating with the apoptosome, and was at fraction correlating with the monomer Apaf-1. [α-33P]dATP after fraction When the nucleotide associated with fractions 10 and was analyzed on a TLC the form of nucleotide in these fractions was [α-33P]dATP (Fig. that dATP was not during apoptosome To measure dATP hydrolysis we incubated [γ-32P]dATP with Apaf-1, Apaf-1 with and Apaf-1 with cytochrome c plus procaspase-9 and measured the released shown in a of hydrolysis was However, the of hydrolysis was not in the presence of cytochromec or cytochrome c plus procaspase-9 with Apaf-1 In dATP binding to Apaf-1 was when cytochrome c and procaspase-9 were present (Fig. In the of dATP hydrolysis was calculated as then 1 We the observed dATP hydrolysis as which is not to Apaf-1 To further confirm that dATP hydrolysis is not for caspase we performed caspase-3 cleavage using nonhydrolyzable ATP and the results with dATP and ATP. shown in Fig. both ATP ATPγS and ADPCP were able to with dATP for Apaf-1 When measured caspase-9 activation directly by the cleavage of its downstream substrate procaspase-3, 1 μm dATP or 100 μm ATP caspase-3 ATPγS to caspase-3 even at 1 a result that was with (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (410) Google Scholar). ADPCP was able to caspase-3 at 100 μm and the activation was with 100 μm ATP when 1 mm ADPCP was ADPCP at 1 mm also apoptosome formation as in Fig. The data a model of caspase activation by Apaf-1. The key event to the binding of dATP or ATP to Apaf-1, which is by the binding of cytochrome c to Apaf-1. The binding of dATP then induces the oligomerization of Apaf-1·cytochromec complex, which procaspase-9 to the The binding of procaspase-9 synergistically dATP binding to Apaf-1. Apaf-1, cytochrome c, and caspase-9 form the holoenzyme that cleaves and activates downstream caspases such as The from the current study was that Apaf-1 alone bound dATP Although Apaf-1 the nucleotide binding that are conserved in the CED-4 protein elegans and the protein these binding not to nucleotides or the binding was so that it not the washing procedure used in the assay. In the presence of cytochrome c, nucleotide binding to Apaf-1 increased Because cytochrome c binding to Apaf-1 in the of nucleotide (3Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar), a with cytochrome c binding to Apaf-1 an event that the nucleotide binding or the binding so that it the washing The was that procaspase-9 further the dATP binding to Apaf-1, the K from to μm (Fig. This that the formation of the holoenzyme of is a event with the binding of caspase-9 to apoptosome further the nucleotide binding or the nucleotide The K of dATP to Apaf-1 in the presence of cytochrome c or cytochromec plus procaspase-9 is well the cytosolic dATP which is 10 μm L. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The nucleotide binding of the C. elegans Apaf-1 homologue CED-4 are also critical for its S. Biol. 1997; Full Text Full Text PDF PubMed Google Scholar). In to Apaf-1, CED-4 protein is the and is in an inactive by the binding of is and CED-4 from the to the region F. D. Y. Science. PubMed Scopus Google Scholar). It is that nucleotide binding to CED-4 regulated with the region of The of the region of CED-4, to its oligomerization and activation of the C. elegans caspase, X. D. Science. 1998; 281: PubMed Scopus Google Scholar). from our and has that the hydrolysis of the of dATP or ATP is for apoptosome formation and caspase activation (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (410) Google Scholar, 8Saleh A. Srinivasula S.M. Acharya S. Fishel R. Alnemri E.S. J. Biol. Chem. 1999; 274: 17941-17945Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). The a direct of ATP hydrolysis by Apaf-1 and the that a nonhydrolyzable ATP analog, was able to caspase activation (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 7Hu Y. Benedict M.A. Ding L. Nunez G. EMBO J. 1999; 18: 3586-3595Crossref PubMed Scopus (410) Google Scholar). In dADP dATP for caspase activation (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar). However, when the Apaf-1 homologue was it was that the critical aspartic acid that are to critical for ATP hydrolysis were to and that ATP hydrolysis not for its A. H. H. P. Wang X. J.M. Biol. 1999; 1: PubMed Scopus Google Scholar). when we the apoptosome and analyzed the nucleotide that bound to the form was dATP, not dADP (Fig. A and B). We that the dATP hydrolysis by Apaf-1 we reported (6Zou H. Li Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar) was from the proteins in the Apaf-1 When we measured the dATP hydrolysis directly in the caspase activation reaction mixture using pure Apaf-1, only the of hydrolysis was and the hydrolysis was not regulated by the presence of cytochrome c and procaspase-9 (Fig. even though we out the that dATP is by Apaf-1, the hydrolysis is and is not correlated with its The of dATP hydrolysis was further out by the that a nonhydrolyzable ATP analog, is sufficient to apoptosome formation and caspase-9 activation (Fig. and ADPCP not the at its position and by Apaf-1. We that the dADP dATP or ATP for caspase-9 activation is that dADP has a for Apaf-1 B). These results also that ATPγS is able to caspase-9 activation not it the of with a it to the oligomerization of Apaf-1 and even Apaf-1 a position so that it with other Apaf-1 The of nucleotide in Apaf-1 is to that of in protein In both it is the form that activates the Apaf-1 and proteins For are that hydrolysis to the and nucleotide that protein activity A. 1994; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). is no that for Apaf-1. of in addition to the of proteins that cytochrome c release and the of proteins that caspase We Li and for We Xie for with the ATP hydrolysis assay and and for the and the