High affinity binding of Bcl-xL to cytochrome c: possible relevance for interception of translocated cytochrome c in apoptosis.
The release of cytochrome c from mitochondria and apoptosis relies on several preferential and selective interactions involving the Bcl-2 family of proteins. There is, however, no direct evidence for the interaction of cytochrome c with these proteins at any stage of apoptosis. To investigate if any pro-survival protein from the Bcl-2 family could intercept cytochrome c after its translocation from mitochondria, the interaction of cytochrome c with bacterially expressed human Bcl-x(L) was studied at pH 7. In size-exclusion chromatography, purified full-length His(6)-tagged Bcl-x(L) migrated as both dimer and monomer, of which the monomeric fractions were used for experiments. Coimmunoprecipitation studies show that cytochrome c interacts with Bcl-x(L). The extent of caspase activity in cell lysate elicited by externally added cytochrome c is reduced when a preincubated mixture of Bcl-x(L) and cytochrome c is used instead. Equilibrium binding monitored by optical absorption of cytochrome c as a function of titrating concentrations of Bcl-x(L) yields the association constant, K(ass) = 8.4(+/- 4) x 10(6) M(-1) (binding affinity, K(diss) = 1/K(ass) approximately 120 nM) which decreases at high ionic strength. The rates for binding of Bcl-x(L) to cytochrome c, studied by stopped-flow kinetics at pH 7, show that the bimolecular rate constant for binding, k(bi) = 0.24 x 10(6) M(-1) s(-1). Values of the thermodynamic and kinetic parameters for Bcl-x(L)-cytochrome c interaction are very similar to those known for regulatory protein-protein interactions in apoptosis.
Yadaiah
M
M
Schools of Chemistry, University of Hyderabad, Hyderabad 500046, India.
Rao
P Nageswara
PN
Harish
P
P
Bhuyan
Abani K
AK
D000818:Animals; D017209:Apoptosis; D045304:Cytochromes c / Q000378:metabolism*; D006736:Horses; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
C494811:BCL2L1 protein, human; D011994:Recombinant Proteins; D051020:bcl-X Protein; D045304:Cytochromes c