Cytochrome c binding to Apaf-1: the effects of dATP and ionic strength.
In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. Here, we show through fluorescence polarization experiments that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c.Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c.Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. We also report here that the cytochrome c.Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins.
Purring-Koch
C
C
Department of Chemistry, Princeton University, Princeton, NJ 08544, USA.
McLendon
G
G
D000818:Animals; D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D006736:Horses; D009994:Osmolar Concentration; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D053477:Apoptotic Protease-Activating Factor 1; D003574:Cytochrome c Group; D003838:Deoxyadenine Nucleotides; D011506:Proteins; D011994:Recombinant Proteins