Abstract
Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK isoenzymes, sarcomeric (sMtCK) and ubiquitous (uMtCK). As compared with human and chicken sMtCK, human uMtCK showed a 23-34 times slower octamer dissociation rate, a reduced reoctamerization rate and a superior octamer stability as deduced from the octamer/dimer ratios at thermodynamic equilibrium. Octamer stability of sMtCK increased with temperature up to 30 degrees C, indicating a substantial contribution of hydrophobic interactions, while it decreased in the case of uMtCK, indicating the presence of additional polar dimer/dimer interactions. These conclusions are consistent with the recently solved x-ray structure of the human uMtCK (Eder, M., Fritz-Wolf, K., Kabsch, W., Wallimann, T., and Schlattner, U. (2000) Proteins 39, 216-225). When binding to 16% cardiolipin membranes, sMtCK showed slightly faster on-rates and higher affinities than uMtCK. However, human uMtCK was able to recruit the highest number of binding sites on the vesicle surface. The observed divergence of ubiquitous and sarcomeric MtCK is discussed with respect to their molecular structures and the possible physiological implications.
Highlights
Mitochondrial creatine kinase (MtCK)1 belongs to the creatine kinase (CK) isoenzyme family occurring in vertebrates and some invertebrate species, especially in tissues with high or fluctuating energy requirements
We found the highest number of bound mitochondrial creatine kinase (MtCK) molecules with human ubiquitous MtCK (uMtCK), followed by chicken sarcomeric MtCK (sMtCK) and human sMtCK (Table II)
Recent evidence strongly suggests that membrane binding and octameric structure of MtCK are essential for the specific functions of the enzyme in the CK/PCr circuit for energy homeostasis [19] and in regulation of the mitochondrial permeability transition pore [8]
Summary
MtCK, mitochondrial creatine kinase; CK, creatine kinase, Cr, creatine; uMtCK and sMtCK, ubiquitous and sarcomeric mitochondrial CK, respectively; PCr, phosphocreatine; PTP, mitochondrial permeability transition pore; TSAC, transition state analogue complex; SPR, surface plasmon resonance; TES, 2-{[2-hydroxy1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. The protective function of MtCK and Cr in Ca2ϩ-induced opening of mitochondrial PTP is abolished [8] if mitochondria are pretreated with the CK transition state analogue complex (TSAC, consisting of ADP, Mg2ϩ, nitrate, and Cr) [20] known to dissociate MtCK octamers. We analyze the transitions of octameric to dimeric MtCK and of free to membrane-bound MtCK in terms of kinetics and thermodynamic equilibrium We compare both uMtCK and sMtCK from human origin, together with the already known chicken sMtCK, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance [22, 25]. It is shown for the first time that ubiquitous and sarcomeric MtCK octamers differ significantly in octamer stability and membrane binding properties
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