Study on heart mitochondrial creatine kinase using a cross-linking bifunctional reagent. I. The binding involves the octameric form of the enzyme

Bovine heart mitochondria suspended in 0.25 M sucrose were treated with 0.3% glutaraldehyde (GA). The membranes were disintegrated by ultrasonication in 0.25 M KCl and precipitated by centrifugation. The supernatant was assayed for creatine kinase (CKm) oligomeric forms by ultracentrifugation in a sucrose density gradient. A kinetic analysis of membrane-bound CKm was performed before and after ultrasonication. The data obtained suggest that the CKm octamer is the only form of CKm bound to mitochondrial membranes during GA treatment. This finding was confirmed by an analysis of extracts from untreated mitochondria using high resolution gel filtration.     

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.     

The quaternary structure of bovine heart mitochondrial creatine kinase

Crosslinking of subunits of the high molecular weight oligomer of bovine heart mitochondrial creatine kinase (CKm) by dimethyl suberimidate and subsequent electrophoresis in the presence of sodium dodecyl sulfate gives eight protein bands. An increase in the time course of the enzyme crosslinking reaction results in the protein accumulation in the high molecular weight bands. Evidence has been obtained suggesting that crosslinking involves only the intraoligomeric contact areas. It is concluded that bovine heart CKm is an octamer. Crosslinking of intersubunit contacts in the octameric form of the enzyme by various diimidates has been carried out. The data obtained suggest that within the octamer the CKm subunits have a quasispherical rather than planar arrangement. This finding is supported by electron microscopy data.     

Interaction of creatine kinase and hexokinase with the mitochondrial membranes,and self-association of creatine kinase: crosslinking studies

Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu⁺⁺-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK Adenylate Kinase (EC 2.7.4.3) - CKm Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2) - DMS Dimethyl Suberimidate - DTT Dithiothreitol - EGS Ethylene Glycol bis (succinimidyl succinate) - EGTA Ethylene Glycol bis (aminoethyl ether) - N,N,N,N Tetraacetic acid - G6P Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid - HK Hexokinase (EC 2.7.1.1) - MABI methyl 4-Azido Benzoimidate - NaPi Sodium Phosphate - SANPAH N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate - SDS Sodium Dodecyl Sulfate (sodium lauryl sulfate) - Tris Tris (hydroxymethyl) Aminomethane     

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.     

Kinetic properties of the octameric and dimeric forms of mitochondrial creatine kinase and physiological role of the enzyme

It has been found that at pH 7.4 and 2 mg/ml protein, bovine heart mitochondrial creatine kinase (CKm) contains less than 10% of the dimer. The constant for the CKm octamer dissociation into dimers, Kd, in the presence of substrates forming an analog of the complex of the transient state was found to be equal to 4.9 10(-17) M3. Using this value, the experimental conditions were chosen so as to achieve a practically complete dissociation of the octamer into dimers. Evidence has been obtained suggesting that the octamer does not dissociate into dimers during the time course of the kinetic experiments; the corresponding kinetic parameters of the CKm octamer and dimer are as follows: KMgATPm = 82 microM and 42 microM; KCrm = 8.1 mM and 3.4 mM; Vf = 61 and 60 micrograms-equiv. H+ min-1 mg-1; KMgADPm = 43 microM and 17 microM, KCPm = 0.68 mM and 0.23 mM; Vr = 162 and 111 micrograms-equiv. H+ min-1 mg-1. The experimental and calculated data shed additional light on the physiological role of CKm.     

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.     

Biochemical effects of PR toxin on rat liver mitochondrial respiration and oxidative phosphorylation

The in vitro effects of PR toxin, a toxic secondary metabolite produced by certain strains of Penicillium roqueforti, on the membrane structure and function of rat liver mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the toxin was added to the assay system. The respiratory control ratio decreased about 60% and the ADP/O ratio decreased about 40% upon addition of 3.1 X 10(-5) M PR toxin to the highly coupled mitochondria. These findings suggest that PR toxin impairs the structural integrity of mitochondrial membranes. On the other hand, the toxin inhibited mitochondrial respiratory functions. It exhibited noncompetitive inhibitions to succinate oxidase, succinate-cytochrome c reductase, and succinate dehydrogenase activities of the mitochondrial respiratory chain. The inhibitory constants of PR toxin to these three enzyme systems were estimated to be 5.1 X 10(-6), 2.4 X 10(-5), and 5.2 X 10(-5) M, respectively. Moreover, PR toxin was found to change the spectral features of succinate-reduced cytochrome b and cytochrome c1 in succinate-cytochrome c reductase and inhibited the electron transfer between the two cytochromes. These observations indicate that the electron transfer function of succinate-cytochrome c reductase was perturbed by the toxin. However, PR toxin did not show significant inhibition of either cytochrome oxidase or NADH dehydrogenase activity of the mitochondria. It is thus concluded that PR toxin exerts its effect on the mitochondrial respiration and oxidative phosphorylation through action on the membrane and the succinate-cytochrome c reductase complex of the mitochondria.     

Hepatic nuclease. 2. Association of polyadenylase, alkaline ribonuclease and deoxyribonuclease with rat-liver mitochondria.

Six types of nuclease activities were found to be concentrated in the large granule fraction isolated from rat liver homogenastes by differential centrifugation. Analysis by density equilibration shows that three nucleases are associated with mitochondria: an alkaline ribonulcease (pH optimum 8.8), an alkaline deoxyribonuclease (pH optimum 7.6) and an enzyme acting on polyriboadenylate (pH optimum 7.5). When the outer mitochondrial membrane is ruptured in hypotonic medium, the three mitochondrial nucleases are partially solubilized. Solubilization is however obtained by addition of KCL to the suspension medium. It is concluded that mitochondrial nucleases are localized in the intermembrane space but that an adsorption to the outer face of the inner mitochondrial membrane occurs in sucrose 0.25 M. The mitochondrial localization of alkaline ribonuclease, alkaline deoxyribonuclease and polyadenylate accounts for at least 80% of the activity of liver homogenate; nevertheless, an excess of these enzymes is present in the microsomal fraction. Although no definite conculusion can be reached for the significance of this observation, it is shown by density equilibration analysis that these nuclease are not associated either with ribosomes or with the membranes which are the major component of the microsomal fraction.     

The effect of lactoperoxidase-catalyzed iodination on the integrity of mitochondrial membranes.

The effect of lactoperoxidase-catalyzed iodination on rat liver mitochondria was investigated. A change from the condensed to the swollen conformation is observed by electron microscopy after extensive iodination of the mitochondria. The outer membrane breaks after incorporation of 0.2 nmol or more iodine atoms per mg of mitochondrial protein releasing adenylate kinase, a soluble enzyme located in the intermembrane space. Further iodination of the mitochondria ruptures the inner membrane, releasing proteins such as glutamic dehydrogenase from the matrix space. Lipid peroxides and I₂ are not intermediates in the disruptive effect of extensive lactoperoxidase-catalyzed iodination on the membranes. During iodination at pH 6.5 almost no release of protein or glutamic dehydrogenase activity is detectable and the loss of adenylate kinase activity from the particulate is diminished. The effect of extensive iodination on mitochondrial membranes limits the amount of iodide which can be incorporated with the lactoperoxidase membrane-labeling procedure when this technique is used as a surface probe of mitochondrial membranes.     

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.     

Determination of isoelectric points of oligomeric forms of mitochondrial creatine kinase

Using isoelectrofocusing in three pH gradients differing in the initial pH value of the ampholyte gel mixture and in gradient pH range, the isoelectric points for the dimeric and octameric forms of mitochondrial creatine kinase from bovine heart and pigeon breast muscle were determined. The isoelectric points for the dimer and octamer are equal to 9.67 +/- 0.01 and 8.93 +/- 0.05 for the heart enzyme and to 9.56 +/- 0.08 and 8.91 +/- 0.23 for the skeletal muscle enzyme. The correctness of identification of the oligomeric forms of mitochondrial creatine kinase was confirmed by ultracentrifugation in a sucrose density linear gradient. Since creatine kinase is known to bind to mitochondrial membrane cardiolipin by electrostatic forces, it can be assumed that both oligomeric forms of the enzymes can bind to the membranes. However, the properties of the creatine kinase dimer suggest its greater ability to bind to mitochondrial membranes.     

Binding of creatine kinase to heart and liver mitochondria in vitro

Phosphate extraction of heart mitochondria results in the release of creatine kinase. Under appropriate conditions phosphate-extracted mitochondria are able to rebind the creatine kinase, either from crude extracts or as the purified enzyme. Heart mitochondria are able to bind up to sevenfold more creatine kinase than they originally contained. The association is specific since the cytoplasmic isozyme from heart (MM) does not bind, and does not interfere with the binding of the mitochondrial isozyme even when MM is present in large excess. It is interesting that although liver mitochondria do not contain the mitochondrial isozyme of creatine kinase they are able to bind approximately the same amount of the enzyme as the heart mitochondria.     

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.     

Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes

Purified mitochondrial creatine kinase (Mi-CK) (EC 2.7.3.2) from chicken heart was shown to interact simultaneously with purified inner and outer mitochondrial membranes, thereby creating an intermembrane chondrial membranes, thereby creating an intermembrane were purified from rat liver and thus were fully devoid of Mi-CK. Intermembrane contact formation was demonstrated by measuring the binding of inner membrane vesicles to outer membranes spread at the air-water interface. Mi-CK also mediated intermembrane adhesion when membranes formed with total lipid extracts of both membranes were used, pointing to the role of lipids as potential membrane anchors of Mi-CK in the mitochondrial intermembrane space. Other enzymes of the intermembrane space that (like Mi-CK) are also cationic, as well as cytosolic isoenzymes of creatine kinase, failed to induce contact formation. Thus, of the proteins tested, membrane contact formation was specific for Mi-CK. The two oligomeric forms of Mi-CK (octamer and dimer) differed in their ability to mediate intermembrane adhesion, the octamer being more potent. Highly basic peptides, i.e. poly-L-lysines, were shown to strongly interact with membranes formed with lipid extracts of mitochondrial membranes: they both induced intermembrane binding and fusion. Interestingly, the extent of contact formation mediated by poly-L-lysines was lower than that of octameric Mi-CK. The implications of these findings on the function and localization of Mi-CK and on the structure of the mitochondrial intermembrane compartment are discussed.     

The structure of mitochondrial creatine kinase and its membrane binding properties

The biochemical and biophysical characterization of the mitochondrial creatine kinase (Mi-CK) from chicken cardiac muscle is reviewed with emphasis on the structure of the octameric oligomer by electron microscopy and on its membrane binding properties. Information about shape, molecular symmetry and dimensions of the Mi-CK octamer, as obtained by different sample preparation techniques in combination with image processing methods, are compared. The organization of the four dimeric subunits into the Mi-CK complex as apparent in the end-on projections is discussed and the consistently observed high binding affinity of the four-fold symmetric end-on faces towards many support films and towards each other is outlined. A study on the oligomeric state of the enzyme in solution and in intact mitochondria, using chemical crosslinking reagents, is presented together with the results of a search for a possible linkage of Mi-CK with the adenine nucleotide translocator (ANT). The nature of Mi-CK binding to model membranes, demonstrating that rather the octameric than the dimeric subspecies is involved in lipid interaction and membrane contact formation, is resumed and put into relation to our structural observations. The findings are discussed in light of a possiblein vivo function of the Mi-CK octamer bridging the gap between outer and inner mitochondrial membranes at the contact sites.     

Creatine kinase of heart mitochondria. The progressive loss of enzyme activity during in vivo ischemia and its correlation to depressed myocardial function

It is now appreciated that mitochondrial creatine kinase (CKm) may play an important role in heart high-energy phosphate metabolism and that this isozyme is solubilized in vitro by dilute solutions of Pi. Since an increase in cellular Pi is known to occur with even brief periods of myocardial ischemia, we investigated the relationship between CKm activity and myocardial performance in rabbit hearts subjected to total global ischemia. CKm activity is expressed as a ratio to mitochondrial malate dehydrogenase (MDHm), a stable marker enzyme. A significant decline in this ratio was observed after only 10 min of ischemia, a time prior to changes in total homogenate creatine kinase activity. After 60 min of ischemia, the CKm/MDHm ratio was depressed by more than 70%. Since there was no restoration of activity following 30 min of reperfusion, we correlated changes in enzyme activity to contractile dysfunction following variable periods of total ischemia. The data showed a close correlation between the decline in the CKm/MDHm ratio and the reduction in performance, measured as left ventricular developed pressure. No correlation was observed between State 3 respiratory rates and performance. Using KCl arrest at 27 degrees C or hyperthermic ischemia at 40 degrees C, the CKm/MDHm ratio consistently correlated to the degree of postischemic functional depression, independent of the duration of ischemia. Isoenzyme electrophoresis failed to detect soluble CKm activity in the postischemic supernatant. Therefore, CKm activity appears to be altered rapidly and irreversibly by ischemia. The implications of these observations on the integration of myocardial high-energy phosphate metabolism are discussed.     

Identification and primary structure of the cardiolipin-binding domain of mitochondrial creatine kinase

It was recently shown that the mitochondrial isozyme of heart creatine kinase binds to cardiolipin on the outer half of the inner membrane [Müller, M., et al. (1985) J. Biol. Chem. 260, 3839-3843]. The enzyme has now been extracted and purified to homogeneity from rat heart mitochondria, and cleaved with CNBr. The fragments have been separated on an FPLC system using a Mono Q HR 5/5 column. Only one of these binds to cardiolipin-containing liposomes and has thus been identified as the cardiolipin-binding domain of the enzyme. Its amino acid sequence has been determined. The fragment contains 25 amino acids and corresponds to the N-terminal region of the protein. The binding of the fragment of cardiolipin-containing liposomes was inhibited by adriamycin. Another and larger CNBr fragment could be specifically labelled with periodate-oxidized (di-aldehyde) ATP and has thus been identified as the ATP-binding domain. Chemical modification of the basic amino acids Lys and Arg of the enzyme abolished its binding to cardiolipin.     

In vitro translation of canine mitochondrial creatine kinase messenger RNA

The cell-free translation products of mRNA from canine myocardium were immunoprecipitated using antiserum specific for either the MM or mitochondrial creatine kinase subunit. The two subunits were shown to be encoded by the nuclear genome and translated from separate mRNAs. The mitochondrial subunit was translated as a polypeptide with a molecular weight approximately 6,000 greater than the mature form of the enzyme. In contrast, the M-subunit was translated as a polypeptide having a molecular weight identical to that of the mature cytosolic M-subunit. It is assumed that the mitochondrial subunit precursor must be proteolytically processed during translocation from the cytoplasm into mitochondria.     

Interaction of mitochondrial creatine kinase with model membranes. A monolayer study

The interaction of mitochondrial creatine kinase (Mi-CK; EC 2.7.3.2) with phospholipid monolayers and spread mitochondrial membranes at the air/water interface has been investigated. It appeared that Mi-CK penetrated into these monolayers as evidenced by an increase in surface pressure upon incorporation of Mi-CK. The increase in surface pressure was dependent on (1) the amount and (2) the oligomeric form of Mi-CK in the subphase, as well as on (3) the initial surface pressure and (4) the phospholipid composition of the monolayer. In this experimental system Mi-CK was able to interact equally well with both inner and outer mitochondrial membranes.