MEMBRANE-BINDING AND LIPID VESICLE CROSS-LINKING KINETICS OF THE MITOCHONDRIAL CREATINE-KINASE OCTAMER

Mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) is a positively char ged enzyme located between the mitochondrial inner and outer membrane as well as along the cristae membranes. The octameric form of Mi-CK is able to cross-link membranes to form contact sites. The process of Mi -CK membrane binding and Mi-CK-induced cross-linking of model membrane vesicles containing different amounts of cardiolipin (CL) was investi gated in vitro. First, the direct binding of octameric Mi-CK to immobi lized lipid vesicles containing cardiolipin was monitored by plasmon r esonance (BiaCore). The analysis of the pseudo-first-order on- and off -rate constants indicates that there are two binding sites with differ ent affinity for Mi-CK on the membrane. The association equilibrium co nstants obtained at 25 degrees C were 813.7 (for 100% CL) and 343.6 (f or 16% CL), respectively, for the high-affinity binding mode. Second, the Mi-CK-induced vesicle cross-linking kinetics were analyzed by fixe d-angle light scattering. Only octameric Mi-CK induced bridged vesicle /protein complexes, whereas dimeric Mi-CK failed to induce vesicle cro ss-linking. For vesicles containing 100% cardiolipin, the pseudo-first -order association rate constant was 2.55 x 10(-3) s(-1), while for me mbranes containing 16% cardiolipin and 84% PC a constant of 6.25 x 10( -3) s(-1) was found. The examined kinetic properties of the system sug gest a two-step model for Mi-CK-induced vesicle cross-linking which co nsists of a fast binding step of the enzyme to the membrane, followed by a remarkably slower cross-linking reaction between Mi-CK-covered ve sicles. The data obtained by in vitro biophysical methods agree with e arlier experiments done with mitoplasts and isolated mitochondrial mem branes and explain the in vivo accumulation of Mi-CK at contact sites between the inner and outer mitochondrial membrane and the formation o f Mi-CK-rich intramitochondrial inclusions observed in creatine-deplet ed animals as well as in patients with mitochondrial cytopathies.