Association of chicken mitochondrial creatine kinase with the inner mitochondrial membrane

The stoichiometry and dissociation constant for the binding of homogeneous chicken heart mitochondrial creatine kinase (MiMi-CK) to mitoplasts was examined under a variety of conditions. Salts and substrates release MiMi-CK from mitoplasts in a manner that suggests an ionic interaction. The binding of MiMi-CK to mitoplasts is competitively inhibited by Adriamycin, suggesting that they compete for the same binding site. Fluorescence measurements also show that Adriamycin binds to MiMi-CK so that the effect of Adriamycin on the binding of MiMi-CK to mitoplasts is not simple. Titrating mitoplasts with homogeneous MiMi-CK at different pH values shows a pH-dependent equilibrium involving a group(s) on either the membrane or the enzyme with a pKa = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/nmol cytochrome aa3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa3. Chicken heart mitochondria contain, after isolation, 2.86 +/- 0.42 IU/nmol cytochrome aa3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ATP translocase/mol cytochrome aa3. Therefore, chicken heart mitoplasts can maximally bind about 1 mol of MiMi-CK per 3 mol translocase; in normal chicken heart mitochondria about 1 mol of MiMi-CK is present per 13 mol translocase.     

Affinity modification of creatine kinase from rabbit skeletal muscle by 2',3'-dialdehyde derivatives of ADP and ATP

Periodate-oxidized ADP and ATP (oADP and oATP) are substrates and affinity reagents for creatine kinase from rabbit skeletal muscle. oADP and oATP modified a lysine epsilon-amino group in the nucleotide-binding site of the enzyme. Complete inactivation is observed upon binding 2 moles oADP per 1 mole of the enzyme dimer. Modification with oADP is described by a liner dependence of the log of enzyme activity on time, testifying to a pseudo-first-order of the reaction. The reaction rate constant (ki = 8.10(3) min-1) and dissociation constant for the reversible enzyme-oADP complex (Kd = 62 microM) were determined. ADP protected the enzyme from inactivation and covalent binding of the analog, whereas oADP covalently bound to the enzyme was phosphorylated by phosphocreatine. The data obtained allow to suggest that the epsilon-amino group of a lysine residue of the active site is located in close proximity to ribose of ATP and ADP forming a complex with the enzyme. This group seems essential for correct orientation of the nucleotide polyphosphate chain in the enzyme active center, but take no immediate part in the transphosphorylation process.     

Determination of molar content of creatine kinase in heart mitochondria by SH-reagents

The maximal content of mitochondrial isoenzyme of creatine kinase (CK) in rat heart mitochondria does not exceed 12.5 moles per mole of ATP-ADP translocase. This value was obtained by titration of mitochondrial CK activity in aged mitochondria by 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) and 2,4-dinitrofluorobenzene (DNFB) and by a more complex and accurate method. The essential thiol groups of membrane-bound mitochondrial CK (and its enzymic activity) can be specifically protected by phosphocreatine (12 mM) + ADP (1-5 mM) against inactivation by DTNB. Mitochondria with protected SH-groups of CK and with groups inactivated by DTNB were repeatedly incubated with DTNB under identical conditions and the number of additionally reacted sulfhydryl groups and the changes in CK activity were measured. The differences in the number of additionally reacted SH-groups correlated with the changes in the CK activity, which made it possible to calculate the molar ratios of mitochondrial CK to cytochrome c oxidase and ATP-ADP translocase (2.16 +/- (0.4): 1:2, respectively).     

Studies of energy transport in heart cells. The functional coupling between mitochondrial creatine phosphokinase and ATP ADP translocase: kinetic evidence.

The dependence of the rate of creatine phosphate synthesis in the mitochondrial creatine phosphokinase reaction upon the rate of oxidative phosphorylation and ATP translocation from the matrix to outside of the mitochondria has been studied. It has been experimentally shown that mitochondrial creatine phosphokinase reacts slowly with ATP in the medium but is very active in utilization of ATP synthesized by the oxidative phosphorylation process. From these data, it is postulated, therefore, that the ATP-ADP translocase transports ATP molecules directly to the active site of creatine phosphokinase localized on the outer site of the inner membrane. This results in an increase in the effective concentration of ATP in the vinicity of the active sites of creatine kinase and in acceleration of the forward reaction (creatine phosphate synthesis). The kinetic theory based on this assumption allows a quantitative explanation of the observed dependences. These data indicate the tight functional coupling between ATP-ADP translocase and creatine phosphokinase in heart mitochondria. It is concluded that in heart cells energy can be transported by creatine phosphate molecules only.     

The lack of direct coupling between ATP-ADP translocase and creatine phosphokinase in isolated rabbit heart mitochondria

The formation of creatine phosphate by isolated rabbit heart mitochondria in the presence of creatine, α-ketoglutarate, ATP, and inorganic phosphate was studied. Creatine phosphate formation was inhibited by oligomycin. This was most probably due to increased concentration of ADP favoring the reverse reaction (formation of creatine and ATP from phosphocreatine and ADP). The inhibitory effect of oligomycin disappeared in the presence of phosphoenolpyruvate and pyruvate kinase. The results do not indicate any direct coupling between mitochondrial creatine phosphokinase and ATP-ADP translocase as has been suggested for rat heart mitochondria.     

Inactivation of beef heart mitochondrial F1-ATPase by the 2',3'-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.     

Influence of mitochondrial creatine kinase on the mitochondrial/extramitochondrial distribution of high energy phosphates in muscle tissue: evidence for a leak in the creatine shuttle

The influence of mitochondrial creatine kinase on subcellular high energy systems has been investigated using isolated rat heart mitochondria, mitoplasts and intact heart and skeletal muscle tissue.In isolated mitochondria, the creatine kinase is functionally coupled to oxidative phosphorylation at active respiratory chain, so that it catalyses the formation of creatine phosphate against its thermodynamic equilibrium. Therefore the mass action ratio is shifted from the equilibrium ratio to lower values. At inhibited respiration, it is close to the equilibrium value, irrespective of the mechanism of the inhibition. The same results were obtained for mitoplasts under conditions where the mitochondrial creatine kinase is still associated with the inner membrane.In intact tissue increasing amounts of creatine phosphate are found in the mitochondrial compartment when respiration and/or muscle work are increased. It is suggested that at high rates of oxidative phosphorylation creatine phosphate is accumulated in the intermembrane space due to the high activity of mitochondrial creatine kinase and the restricted permeability of reactants into the extramitochondrial space. A certain amount of this creatine phosphate leaks into the mitochondrial matrix.This leak is confirmed in isolated rat heart mitochondria where creatine phosphate is taken up when it is generated by the mitochondrial creatine kinase reaction. At inhibited creatine kinase, external creatine phosphate is not taken up. Likewise, mitoplasts only take up creatine phosphate when creatine kinase is still associated with the inner membrane. Both findings indicate that uptake is dependent on the functional active creatine kinase coupled to oxidative phosphorylation.Creatine phosphate uptake into mitochondria is inhibited with carboxyatractyloside. This suggests a possible role of the mitochondrial adenine nucleotide translocase in creatine phosphate uptake.Taken together, our findings are in agreement with the proposal that creatine kinase operates in the intermembrane space as a functional unit with the adenine nucleotide translocase in the inner membrane for optimal transfer of energy from the electron transport chain to extramitochondrial ATP-consuming reactions.     

Heart mitochondria in physiological salt solution: not ionic strength but salt composition is important for association of creatine kinase with the inner membrane surface

In physiological salt solution (PSS) which mimicks the cardiac cells cytoplasm and contains 120 mM K-MES, 10 mM NaCl, 20 mM imidazole, pH 7.2, 20 mM taurine, 15 mM creatine, 15 mM Na2phosphocreatine, 5 mM Na2ATP, 8 mM MgCl2, 5 mM K2HPO4, 3 mM glutamate, 3 mM malate, 0.5 mM dithiothreitol and 10 mg/ml of bovine serum albumine both isolated mitochondria and intracellular structures in skinned fibers stay intact. In PSS mitochondrial creatine kinase remains firmly attached to the inner membrane surface. CKmi-mi is extracted from cardiac mitoplasts in 0.125 M KCl solution, but addition of 10 mM sodium borate to this KCl solution completely inhibits dissociation of CKmi-mi. Therefore, not ionic strength but ion composition is important for association of CKmi-mi with mitochondrial membrane. Functional and structural studies using antibodies against CKmi-mi showed that in PSS CKmi-mi is bound to the inner mitochondrial membrane in spatially close relationship to adenine nucleotide translocase (ANT). Thus, under physiological conditions CKmi-mi is structurally and functionally coupled to ANT in cardiac mitochondria and functions to catalyze almost complete utilization of mitochondrial ATP for aerobic phosphocreatine synthesis.     

Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions.

1. The kinetic properties of mitochondrial creatine phosphokinase (Km for all substrates and maximal rates of the forward and reverse reaction) have been studied. Since (a) Km value for MgADP- (0.05 mM) and creatine phosphate (0.5 mM) are significantly lower than Km for MgATP2- (0.7 mM) and creatine (5.0 mM) and (b) maximal rate of the reverse reaction (creatine phosphate + ADP leads to ATP + creatine) equal to 3.5 mumol times min-1 times mg-1 is essentially higher than maximal rate of the forward reaction (0.8 mumol times min-1 times mg-1), ATP synthesis from ADP and creatine phosphate is kinetically preferable over the forward reaction. 2. A possible regulatory role of Mg2+ ions in the creatine phosphokinase reaction has been tested. It has been shown that in the presence of all substrates and products of the reaction the ratio of the rates of forward and reverse reactions can be effectively regulated by the concentration of Mg2+ ions. At limited Mg2+ concentrations creatine phosphate is preferably synthesized while at high Mg2+ concentrations (more ATP in the reaction medium) ATP synthesis takes place. 3. The kinetic (mathematical) model of the mitochondrial creatine phosphokinase reaction has been developed. This model accounts for the existence of a variety of molecular forms of adenine nucleotides in solution and the formation of their complexes with magnesium. It is based on the assumption that the mitochondrial creatine phosphokinase reactions mechanism is analogous to that for soluble isoenzymes. 4. The dependence of the overall rate of the creatine phosphokinase reaction on the concentration of total Mg2+ ions calculated from the kinetic model quantitatively correlates with the experimentally determined dependence through a wide range of substrates (ATP, ADP, creatine and creatine phosphate) concentration. The analysis of the kinetic model demonstrates that the observed regulatory effect of Mg2+ on the overall reaction rate can be expained by (a) the sigmoidal variation in the concentration of the MgADP- complex resulting from the competition between ATP AND ADP for Mg2+ and (b) the high affinity of the enzyme to MgADP-. 5. The results predicted by the model for the behavior of mitochondrial creatine phosphokinase under conditions of oxidative phosphorylation point to an intimate functional interaction of mitochondrial creatine phosphokinase and ATP-ADP translocase.     

Compartmented coupling of chicken heart mitochondrial creatine kinase to the nucleotide translocase requires the outer mitochondrial membrane

The kinetic coupling of mitochondrial creatine kinase (MiMi-CK) to ADP/ATP translocase in chicken heart mitochondrial preparations is demonstrated. Measuring the MiMi-CK apparent Km value for MgATP2- (at saturating creatine) gives a value of 36 microM when MiMi-CK is coupled to oxidative phosphorylation. This Km value is threefold lower than the Km for enzyme bound to mitoplasts or free in solution. The nucleotide translocase Km value for ADP decreases from 20 to 10 microM in the presence of 50 mM creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased Km values. The observed Km differences can be used to calculate the concentration of ATP and ADP under steady-state conditions showing that the observed differences in the kinetic constants accurately reflect the enzyme activities of MiMi-CK under the different conditions. The behavior of the Km values provides evidence for what we term compartmented coupling. Therefore, like the rabbit heart system (S. Erickson-Viitanen, P. Viitanen, P. J. Geiger, W. C. T. Yang, and S. P. Bessman (1982) J. Biol. Chem. 257, 14395-14404) compartmented coupling requires an intact outer mitochondrial membrane. The apparent Km values for normal or compartmentally coupled systems can be used to calculate steady-state values of ATP and ADP by coupling enzyme theory. Hence, the overall kinetic parameters accurately reflect the behavior of the enzymes whether free in solution or in the intermembrane space.     

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

Decreased mitochondrial creatine kinase activity in dystrophic chicken breast muscle alters creatine-linked respiratory coupling

Dystrophic chicken breast muscle mitochondria contain significantly less mitochondrial creatine kinase than normal breast muscle mitochondria. Breast muscle mitochondria from normal 16- to 40-day-old chickens contain approximately 80 units of mitochondrial creatine kinase per unit of succinate:INT (p-iodonitrotetrazolium violet) reductase, a mitochondrial marker, while dystrophic chicken breast muscle mitochondria contain 36-44 units. Normal chicken heart muscle mitochondria contain about 10% of the mitochondrial creatine kinase per unit of succinate:INT reductase as normal breast muscle mitochondria. The levels in heart muscle mitochondria from dystrophic chickens are not affected significantly. Evidence is presented which shows that the reduced level of mitochondrial creatine kinase in dystrophic breast muscle mitochondria is responsible for an altered creatine linked respiration. First, both normal and dystrophic breast muscle mitochondria respire with the same state 3 and state 4 respiration. Second, the post-ADP state 4 rate of respiration of normal breast muscle mitochondria in the presence of 20 mM creatine continues at the state 3 rate. However, the state 4 rate of dystrophic breast muscle mitochondria and mitochondria from other muscle types with a low level of mitochondrial creatine kinase, such as heart muscle and 5-day-old chicken breast muscle, is slower than the state 3 rate. Third, dystrophic breast mitochondria synthesize ATP at the same rate as normal breast muscle mitochondria but rates of creatine phosphate synthesis in 20-50 mM Pi are reduced significantly. Finally, increasing concentrations of Pi displace mitochondrial creatine kinase from mitoplasts of normal and dystrophic breast muscle mitochondria with the same apparent KD, indicating that the outer surface of the inner mitochondrial membrane and the mitochondrial creatine kinase from dystrophic muscle are not altered.     

Mathematical modeling of intracellular transport processes and the creatine kinase systems: a probability approach

A probability approach was used to describe mitochondrial respiration in the presence of substrates, ATP, ADP, Cr and PCr. Respiring mitochondria were considered as a three-component system, including: 1) oxidative phosphorylation reactions which provide stable ATP and ADP concentrations in the mitochondrial matrix; 2) adenine nucleotide translocase provides exchange transfer of matrix adenine nucleotides for those from outside, supplied from medium and by creatine kinase; 3) creatine kinase, starting these reactions when activated by the substrates from medium. The specific feature of this system is close proximity of creatine kinase and translocase molecules. This results in high probability of direct activations of translocase by creatine kinase-derived ADP or ATP without their leak into the medium. In turn, the activated translocase with the same high probability directly provides creatine kinase with matrix-derived ATP or ADP. The catalytic complexes of creatine kinase formed with ATP from matrix together with those formed from medium ATP provide activation of the forward creatine kinase reaction coupled to translocase activation. Simultaneously the catalytic complexes of creatine kinase formed with ADP from matrix together with those formed from medium ADP provide activation of the reverse creatine kinase reaction coupled to translocase activation. The considered probabilities were arranged into a mathermatical model. The model satisfactorily simulates the available experimental data by several groups of investigators. The results allow to consider the observed kinetic and thermodynamic iriegularities in behavior of structurally bound creatine kinase as a direct consequence of its tight coupling to translocase.     

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.     

Inhibition of the mitochondrial permeability transition by creatine kinase substrates. Requirement for microcompartmentation

Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by beta-guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.     

Affinity labeling of purified Ca2+,Mg2+-activated ATPase of Escherichia coli by the 2',3'-dialdehydes of adenosine 5'-di- and triphosphates

The 2′,3′-dialdehydes of ADP and ATP (oADP and oATP), obtained by periodate oxidation of ADP and ATP, inhibited the hydrolytic activity of the purified Ca²⁺.Mg²⁺-activated ATPase of Escherichia coli. Nonspecific labeling of amino groups by these dialdehydes was corrected by carrying out the reactions in the presence of 15 mm ATP. Two types of modification of “ATP-protectable” binding sites by oATP could be detected. The binding of 2 mol “ATP-protectable” oATP/mol ATPase was without affect on ATPase activity and still occurred in the hydrolytically inactive ATPase of an unc A mutant. The binding of a further 3 mol “ATP-protectable” oATP/mol ATPase resulted in almost complete loss of ATPase activity although much of the loss occurred during the binding of the first additional molecule of oATP. This additional ATP-protectable oATP binding did not occur in the unc A mutant and so resembled both the inhibitory effect of oADP on the ATPase activity of normal strains and its lack of binding to the unc A ATPase (P. D. Bragg and C. Hou, 1980, Biochem. Biophys. Res. Commun.95, 952–957). The “ATP-protectable” binding sites for oADP and oATP were located on the α subunit of the ATPase. Binding of oADP or oATP did not result in release of the tightly bound ADP and ATP from the enzyme. We conclude that separate binding sites for oADP and oATP occur on the α subunits of the E. coli ATPase and that the former may be the active site(s) for ATP hydrolysis while the latter are involved in regulation of the ATPase complex.     

Heart mitochondrial creatine kinase revisited: the outer mitochondrial membrane is not important for coupling of phosphocreatine production to oxidative phosphorylation

The state of mitochondrial creatine kinase (CKmi-mi) in intact dog heart mitochondria and mitoplasts and the mechanism of its functional coupling with the oxidative phosphorylation system have been reinvestigated under different osmotic conditions and ionic compositions of the medium. It has been established that in a medium which mimics the cardiac cell cytoplasma, dissociation of CKmi-mi from the membrane of mitoplasts increases when the mitoplasts are swollen due to hypoosmotic treatment. It was shown by EPR that hypoosmotic treatment results in the enhancement of the mobility of phospholipids in the membrane bilayer. It has been also shown that when CKmi-mi is detached from the inner membrane in intact mitochondria in isotonic KCl solution, the effects of the coupling between CKmi-mi and oxidative phosphorylation via ATP/ADP translocase disappear in spite of the presence of CKmi-mi in the intermembrane space and intactness of the outer mitochondrial membrane. Therefore, this coupling cannot be explained by the "compartmented coupling" mechanism or "dynamic adenine nucleotide compartmentation" in the intermembrane space due to diffusion limitation for adenine nucleotides through the outer mitochondrial membrane, as has been supposed by several authors (F.N. Gellerich et al. (1987) Biochim. Biophys. Acta 890, 117-126; S.P.J. Brooks and C.H. Suelter (1987) Arch. Biochem. Biophys. 253, 122-132). The data obtained show that the displacement of the enzyme from the membrane results in significantly increased sensitivity of the coupled processes of aerobic phosphocreatine synthesis to inhibition by the product, phosphocreatine. Thus, all results show that under physiological osmotic and ionic conditions CKmi-mi remains firmly attached to the inner mitochondrial membrane and effectively coupled with ATP/ADP translocase due to intimate dynamic interaction between those proteins.     

Specific inhibition of ATP-ADP translocase in cardiac mitoplasts by antibodies against mitochondrial creatine kinase

Mitochondrial creatine kinase was purified from rat hearts and used to produce antibodies in chicken and rabbits. Antibodies were purified to a high degree of homogeneity by an affinity chromatography method. Chicken antibodies against mitochondrial creatine kinase inhibited this enzyme in rat-heart mitochondrial inner membrane and matrix preparation, and simultaneously blocked oxidative phosphorylation. Under these conditions respiratory chain activities remained unchanged, but adenine nucleotide translocase was inhibited. Removal of mitochondrial creatine kinase from the membrane by pretreatment with 0.15 M KCl and 20 mM ADP completely abolished the effect of antibodies against mitochondrial creatine kinase on oxidative phosphorylation. Noninhibitory antibodies from rabbit with high affinity to rat mitochondrial creatine kinase inhibited neither creatine kinase activity nor oxidative phosphorylation. These data show close and specific spatial arrangement of mitochondrial creatine kinase and adenine nucleotide translocase in mitochondria. It is supposed that there is a fixed orientation of these proteins in the cardiolipin domain in the membrane and that their interaction may occur by a frequent collision due to their lateral movement.     

Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage

The creatine/phosphocreatine circuit provides an efficient energy buffering and transport system in a variety of cells with high and fluctuating energy requirements. It connects sites of energy production (mitochondria, glycolysis) with sites of energy consumption (various cellular ATPases). The cellular creatine/phosphocreatine pool is linked to the ATP/ADP pool by the action of different isoforms of creatine kinase located at distinct subcellular compartments. Octameric mitochondrial creatine kinase (MtCK), together with porin and adenine nucleotide translocase, forms a microcompartment at contact sites between inner and outer mitochondrial membranes and facilitates the production and export into the cytosol of phosphocreatine. MtCK is probably in direct protein-protein contact with outer membrane porin, whereas interaction with inner membrane adenine nucleotide translocase is rather mediated by acidic phopholipids (like cardiolipin) present in significant amounts in the inner membrane. Octamer-dimer transitions of MtCK as well as different creatine kinase substrates have a profound influence on controlling mitochondrial permeability transition (MPT). Inactivation by reactive oxygen species of MtCK and destabilization of its octameric structure are factors that contribute to impairment of energy homeostasis and facilitated opening of the MPT pore, which eventually lead to tissue damage during periods of ischemia/reperfusion.     

Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase

The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.