Christian Vial

Only one of the two interconvertible forms of mitochondrial creatine kinase binds to heart mitoplasts

When analyzed by cellulose acetate electrophoresis, solubilized pig or rabbit heart mitochondrial creatine kinase is shown to exist under two distinct forms. The less cathodic one (form 1) is a dimer and the other having a higher cathodic mobility (form 2) has a molecular weight of about 350,000. The latter form can be converted into the former by incubation at alkaline pH or when the enzyme forms a reactive or an abortive complex with its substrates. This conversion is a reversible phenomenon and is not due to proteolysis. When rabbit heart mitoplasts are treated with the creatine kinase releasing agents, the enzyme is always solubilized as its form 2 and conversion to form 1, when it occurs, always take place after solubilization. Form 2 is also the only form which can be bound to pig or rabbit mitoplasts. Thus form 2 may be the actual form associated with heart mitochondria in vivo.

Role of quaternary structure in muscle creatine kinase stability: tryptophan 210 is important for dimer cohesion.

A mutant of the dimeric rabbit muscle creatine kinase (MM‐CK) in which tryptophan 210 was replaced has been studied to assess the role of this residue in dimer cohesion and the importance of the dimeric state for the native enzyme stability. Wild‐type protein equilibrium unfolding induced by guanidine hydrochloride occurs through intermediate states with formation of a molten globule and a premolten globule. Unlike the wild‐type enzyme, the mutant inactivates at lower denaturant concentration and the loss of enzymatic activity is accompanied by the dissociation of the dimer into two apparently compact monomers. However, the Stokes radius of the monomer increases with denaturant concentration as determined by size exclusion chromatography, indicating that, upon monomerization, the protein structure is destabilized. Binding of 8‐anilinonaphthalene‐1‐sulfonate shows that the dissociated monomer exposes hydrophobic patches at its surface, suggesting that it could be a molten globule. At higher denaturant concentrations, both wild‐type and mutant follow similar denaturation pathways with formation of a premolten globule around 1.5‐M guanidine, indicating that tryptophan 210 does not contribute to a large extent to the monomer conformational stability, which may be ensured in the dimeric state through quaternary interactions. Proteins 32:43–51, 1998.

Immunological determination of the oligomeric form of mitochondrial creatine kinase in situ

Whereas factors governing the interconversion of the two oligomeric forms of mitochondrial creatine kinase are relatively well known, few informations are yet available on the actual form in situ. Antibodies against purified pig and rabbit heart mitochondrial creatine kinase were obtained. The former exhibits a marked specificity for the dimer while the second reacts with both dimer and octamer. They allowed to demonstrate that no dimer can be detected in mitochondria and that CKm occurs naturally exclusively as an octamer. We present arguments that the larger part, if not the totality, of the octamer is membrane‐bound rather than soluble in the intennembrane space. However, these findings do not refute the previously proposed models for the regulation of CKm activity in the mitochondrion but urge to envisage a more complex one.

Proteinase K Processing of Rabbit Muscle Creatine Kinase

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328–K380). However, the C-terminal end of the K1 fragment is not A327 as expected, but D325. Thus, the amino acids residues T326 and A327 have been eliminated by the protease.

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the “protein decorated micelle structure” or the “necklace structure.”

Mg-nucleotides induced dissociation of liposome-bound creatine kinase: reversible changes in its secondary structure and in the fluidity of the bilayer

MgADP binding to mitochondrial creatine kinase (mtCK) adsorbed on liposomes was induced by the photorelease of caged ADP. The nucleotide binding produced two types of structural changes. One was related to the well-established release of mtCK from the liposomes. The other corresponded to reversible structural changes induced by nucleotide binding to mtCK as demonstrated here. Infrared spectroscopy data show that the MgADP-induced desorption of mtCK from vesicles led to a slight increase in f -helix structures in mtCK at the expense of a small decrease in g -sheet structures and a concomitant increase in the fluidity of the membranes. The desorption of mtCK induced by MgADP and MgATP was almost complete, as shown by centrifugation and enzymatic activity measurements. The photorelease of MgADP in a reactive medium containing phosphocreatine and mtCK associated with liposomes led to nucleotide binding and to the formation of MgATP and creatine. Addition of phosphocreatine also desorbed mtCK from liposomes, while addition of creatine did not. Interpretation of these results would suggest that ADP, ATP or phosphocreatine induce the release of mtCK from membranes, increase the phospholipid bilayer fluidity, and may also decrease the number of contact sites between inner and outer mitochondrial membranes, thus affecting the activity of other mitochondrial enzymes. It is tempting to propose that membrane mtCK binding regulation by nucleotide and PCr concentrations may serve as a physiological adaptation for energy supply.

The Normal and Pathological Structure, Function and Expression of Mitochondrial Creatine Kinase

Creatine kinase (CK) isoenzymes are expressed in tissues with important and rapid energy requirements. These enzymes catalyse the reversible phosphorylation of creatine by ATP.