cytochrome-c-t and 6-carboxyfluorescein

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H2O2, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H2O2-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H2O2-induced liposome leakage was abolished by cyanide presumably competing with H2O2 for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H2O2 permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H2O2, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.

Topics: Algorithms; Animals; Antioxidants; Butylated Hydroxytoluene; Cardiolipins; Chromans; Cytochromes c; Dextrans; Fluoresceins; Hydrogen Peroxide; Lipid Peroxidation; Liposomes; Models, Chemical; Models, Molecular; Oxidants; Permeability; Protein Binding; Quercetin; Rhodamines; Spectrometry, Fluorescence

The interaction between cytochrome c and its heme-free precursor apocytochrome c and chemically prepared fragments of these basic proteins with phosphatidylserine containing model membrane systems was studied by differential scanning calorimetry and carboxyfluorescein release experiments. Addition of apocytochrome c and fragments derived from the N-terminus cause a pronounced and linear decrease of the enthalpy (delta H) of the gel to liquid-crystalline phase transition of dielaidoylphosphatidylserine. In contrast, fragments derived from the C-terminus cause a smaller reduction in delta H; a similar trend was observed for the ability of the fragments to cause an increased carboxyfluorescein release from unilamellar vesicles. In addition, the covalent attachment of the heme at cysteine residues 14 and 17 greatly reduced the ability of both the intact protein and the N-terminal fragments to decrease delta H. Using a protein translocation assay based on large unilamellar vesicles containing enclosed trypsin it was found that at gel state temperatures the ability of apocytochrome c to partially translocate the bilayer (reach the opposite membrane/water interface) was greatly reduced. The implications of these findings for the import mechanism of apocytochrome c in mitochondria are shortly indicated.

Topics: Animals; Apoproteins; Biological Transport; Calorimetry, Differential Scanning; Chemical Phenomena; Chemistry, Physical; Cytochrome c Group; Cytochromes c; Fluoresceins; Gels; Heme; Horses; Membrane Lipids; Models, Biological; Phospholipids; Protein Binding; Structure-Activity Relationship; Thermodynamics