Association of chicken mitochondrial creatine kinase with the inner mitochondrial membrane

Abstract

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a p K a = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/ nmol cytochrome aa 3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa 3. Chicken heart mitochondria contain, after isolation, 2.86 ± 0.42 IU/nmol cytochrome aa 3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ ATP translocase/mol cytochrome aa 3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.