Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships

The mitochondrial isoform of creatine kinase (Mi-CK, EC 2.7.3.2) purified to homogeneity from chicken cardiac muscle by the mild and efficient technique described in this article was greater than or equal to 99.5% pure and consisted of greater than or equal to 95% of a distinct, octameric Mi-CK protein species, with a Mr of 364,000 +/- 30,000 and an apparent subunit Mr of 42,000. The remaining 5% were dimeric Mi-CK with an apparent Mr of 86,000 +/- 8,000. Octamerization was not due to covalent linkages or intermolecular disulfide bonding. Upon dilution into buffers of low ionic strength and alkaline pH, octameric Mi-CK slowly dissociated in a time-dependent manner (weeks-months) into dimeric Mi-CK. However, the time scale of dimerization was reduced to minutes by the addition to diluted Mi-CK octamers of a mixture of Mg2+, ADP, creatine and nitrate known to induce a transition-state analogue complex (Milner-White, E.J., and Watts, D. C. (1971) Biochem. J. 122, 727-740). The conversion was fully reversible, and octamers were reformed by simple concentrations of Mi-CK dimer solutions to greater than or equal to 1 mg/ml at near neutral pH and physiological salt concentrations in the absence of adenine nucleotide. After separation of the two Mi-CK species by gel filtration, electron microscopic analysis revealed uniform square-shaped particles with a central negative-stain-filled cavity in the octamer fractions and “banana-shaped” structures in the dimer fractions. Mi-CK was localized inside the mitochondria by immunogold labeling with polyclonal antibodies. A dynamic model of the octamer-dimer equilibrium of Mi-CK and the preferential association of the octameric Mi-CK form with the inner mitochondrial membrane is discussed in the context of regulation of Mi-CK activity, mitochondrial respiration, and the CP shuttle.