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  • Title: Membrane-binding and lipid vesicle cross-linking kinetics of the mitochondrial creatine kinase octamer.
    Author: Stachowiak O, Dolder M, Wallimann T.
    Journal: Biochemistry; 1996 Dec 03; 35(48):15522-8. PubMed ID: 8952506.
    Abstract:
    Mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) is a positively charged 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 investigated in vitro. First, the direct binding of octameric Mi-CK to immobilized lipid vesicles containing cardiolipin was monitored by plasmon resonance (BiaCore). The analysis of the pseudo-first-order on- and off-rate constants indicates that there are two binding sites with different affinity for Mi-CK on the membrane. The association equilibrium constants obtained at 25 degrees C were 813.7 (for 100% CL) and 343.6 (for 16% CL), respectively, for the high-affinity binding mode. Second, the Mi-CK-induced vesicle cross-linking kinetics were analyzed by fixed-angle light scattering. Only octameric Mi-CK induced bridged vesicle/protein complexes, whereas dimeric Mi-CK failed to induce vesicle cross-linking. For vesicles containing 100% cardiolipin, the pseudo-first-order association rate constant was 2.55 x 10(-3) s-1, while for membranes 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 suggest a two-step model for Mi-CK-induced vesicle cross-linking which consists of a fast binding step of the enzyme to the membrane, followed by a remarkably slower cross-linking reaction between Mi-CK-covered vesicles. The data obtained by in vitro biophysical methods agree with earlier experiments done with mitoplasts and isolated mitochondrial membranes and explain the in vivo accumulation of Mi-CK at contact sites between the inner and outer mitochondrial membrane and the formation of Mi-CK-rich intramitochondrial inclusions observed in creatine-depleted animals as well as in patients with mitochondrial cytopathies.
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