Heart Mitochondrial Creatine Kinase Research Articles

Cloning, Escherichia coli expression, and phase-transition chromatography-based purification of recombinant rabbit heart mitochondrial creatine kinase.

A cDNA clone of the mitochondrial sarcomeric creatine kinase cDNA was obtained by screening a rabbit heart library. This cDNA is characterized by a 1257-nucleotide open reading frame encoding a 419-amino-acid protein with a cleavable 39-amino-acid mitochondrial presequence (Accession No. AJ011334). This new member of the guanidino kinase family shows a high degree of sequence similarity with the other phosphagen kinases sequenced so far. The mature enzyme was efficiently expressed in Escherichia coli BL21(DE3) cells as a soluble octameric protein using the pET21 plasmid and purified by a three-step improved method including a final phase-transition chromatography.

Effects of γ-glutamylcysteine ethyl ester on heart mitochondrial creatine kinase activity: involvement of sulfhydryl groups

To study the mechanism of the protective effect of γ-glutamylcysteine ethyl ester, mitochondrial creatine kinase activity of rat heart was measured. γ-Glutamylcysteine ethyl ester had a protective effect against the depression of creatine kinase activity induced by xanthine+xanthine oxidase or hydrogen peroxide. γ-Glutamylcysteine ethyl ester also prevented the depression of creatine kinase activity induced by N-ethylmaleimide. It is suggested that the protective effect of γ-glutamylcysteine ethyl ester is related to oxygen free radicals or to reduction of the sulfhydryl groups of the enzyme which were previously oxidized by oxygen free radicals.

Adriamycin-Fe(3+)-induced inactivation of rat heart mitochondrial creatine kinase: sensitivity to lipid peroxidation.

Adriamycin (ADM)-Fe3+ caused inactivation of rat heart mitochondrial creatine kinase (CK) with lipid peroxidation. Superoxide dismutase, catalase and hydroxyl radical scavengers were without effect on the CK inactivation and the lipid peroxidation induced by ADM-Fe3+, indicating the lack of involvement of superoxide, hydrogen peroxide or hydroxyl radicals in these reactions. The antioxidant butylated hydroxytoluene strongly inhibited not only lipid peroxidation but also CK inactivation, indicating that mitochondrial CK was inactivated with lipid peroxidation. Reduced glutathione and dithiothreitol (DTT) prevented CK inactivation without inhibiting lipid peroxidation. The CK activity of 5,5'-dithiobis-(nitorobenzoic acid)-treated mitochondria exposed to ADM-Fe3+ was partially reversed by addition of DTT, indicating that CK inactivation was due to oxidative damage of sulfhydryl groups. In contrast, mitochondrial protein SH groups were not attacked via ADM-Fe(3+)-induced lipid peroxidation. Thus, the SH groups in mitochondrial CK are very susceptible to ADM-Fe(3+)-induced lipid peroxidation.

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.

Effects of Corn Oil Addition to the Diet on the Energy Metabolism of Heart, Liver and Kidney of Female Rats

Female rats (SIV-50, Sprague Dawley) given a diet enriched with corn oil (10% and 25% addition to rat chow, w/w) for four weeks showed changes in the energy metabolism of heart, liver and kidneys, that is, changes in oxygen consumption and uncoupling of oxidative phosphorylation of heart, liver and kidney mitochondria, concomitant to a decrease in heart mitochondrial creatine kinase activity and an increase in heart mitochondrial creatine content. The high fat diet also affected calcium binding and Na/K-, Mg- and Ca-ATPases of a cardiac myocyte membrane fraction. Lipid feeding also led to increase (biphasic and transient) of phospholipids, triglycerides and free fatty acids in the three organs studied, but in no case to hypertrophy.

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.

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 cytoplasm, 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.

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 K m value for MgATP 2− (at saturating creatine) gives a value of 36 μ m when MiMi-CK is coupled to oxidative phosphorylation. This K m value is threefold lower than the K m for enzyme bound to mitoplasts or free in solution. The nucleotide translocase K m value for ADP decreases from 20 to 10 μ m in the presence of 50 m m creatine only with intact mitochondria. Similar experiments with mitoplasts do not give decreased K m values. The observed K m 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 K m 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 K m 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.

Homogeneous chicken heart mitochondrial creatine kinase purified by dye-ligand and transition-state analog-affinity chromatography

A method for the preparation of homogeneous mitochondrial creatine kinase from chicken heart is presented. The two-column procedure, which can be completed in 2 days, uses Procion red dye and transition-state analog-affinity chromatography. The transition-state analog-affinity chromatographic system utilizes an ADP-hexane-agarose column in conjunction with the transition-state analog complex originally developed by E. J. Milner-White and D. C. Watts (1971, Biochem. J. 122, 727–740) composed of KNO 3, MgCl 2, creatine, and ADP. The enzyme is a dimer composed of 2 M r 43,000 subunits. The sequence of the first N-terminal 20 amino acids shows that the enzyme is different from the cytosolic isozymes but similar to human mitochondrial creatine kinase. The enzyme has an extinction coefficient of ϵ 280 nm = 2.22 ± 0.10 ml·mg −1·cm −1 and a maximum velocity of 200 IU/ml at pH 7.0. The kinetic constants for the chicken heart mitochondrial isozyme are comparable to values for the canine and human heart isozyme.

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 p K a = 6. Extrapolating these titrations to infinite MiMi-CK concentration gives 14.6 IU bound/ nmol cytochrome aa 3 corresponding to 1.12 mol MiMi-CK/mol cytochrome aa 3. Chicken heart mitochondria contain, after isolation, 2.86 ± 0.42 IU/nmol cytochrome aa 3. Titrating respiring mitoplasts with carboxyatractyloside gives at saturation 3.3 mol ADP/ ATP translocase/mol cytochrome aa 3. 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.

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.

Creatine kinase of heart mitochondria. Control of oxidative phosphorylation by the extramitochondrial concentrations of creatine and phosphocreatine.

Defining how extramitochondrial high-energy phosphate acceptors influence the rates of heart oxidative phosphorylation is essential for understanding the control of myocardial respiration. When the production of phosphocreatine is coupled to electron transport via mitochondrial creatine kinase, the net reaction can be expressed by the balanced equation: creatine + Pi----phosphocreatine + H2O. This suggests that rates of oxygen consumption could be regulated by changes in [creatine], [Pi], or [phosphocreatine], alone or in combination. The effects of altering these metabolites upon mitochondrial rates of respiration were examined in vitro. Rat heart mitochondria were incubated in succinate-containing oxygraph medium (pH 7.2, 37 degrees C) supplemented with five combinations of creatine (1.0-20 mM), phosphocreatine (0-25 mM), and Pi (0.25-5.0 mM). In all cases, the mitochondrial creatine kinase reaction was initiated by additions of 0.5 mM ATP. To emphasize the duality of control, the results are presented as three-dimensional stereoscopic projections. Under physiological conditions, with 5.0 mM creatine, increases in Pi or decreases in phosphocreatine had little influence upon mitochondrial respiration. When phosphocreatine was held constant (15 mM), changes in [creatine] modestly stimulated respiratory rates, whereas Pi again showed little effect. With 1.0 mM Pi, respiration clearly became dependent upon changes in [creatine] and [phosphocreatine]. Initially, respiratory rates increased as a function of [creatine]. However, at [phosphocreatine] values below 10 mM, product "deinhibition" was observed, and respiratory rates rapidly increased to 80% State 3. With 2.0 mM Pi or higher, respiration could be regulated from State 4 to 100% State 3. Overall, the data show how increasing [creatine] and decreasing [phosphocreatine] influence the rates of oxidative phosphorylation when mediated by mitochondrial creatine kinase. Thus, these changes may become secondary cytoplasmic signals regulating heart oxygen consumption.

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.

Creatine kinase of heart mitochondria: Changes in its kinetic properties induced by coupling to oxidative phosphorylation

A complete kinetic analysis of the forward mitochondrial creatine kinase reaction was conducted to define the mechanism for its rate enhancement when coupled to oxidative phosphorylation. Two experimental systems were employed. In the first, ATP was produced by oxidative phosphorylation. In the second, heart mitochondria were pretreated with rotenone and oligomycin, and ATP was regenerated by a phosphoenolpyruvate-pyruvate kinase system. Product inhibition studies showed that oxidative phosphorylation did not effect the binding of creatine phosphate to the enzyme. Creatine phosphate interacted competitively with both ATP and creatine, and the E · MgATP · CrP dead-end complex was not readily detected. In a similar manner, the dissociation constants for creatine were not influenced by the source of ATP: K ib = 29 mm; K b = 5.3 mM, and the maximum velocity of the reaction was unchanged: V 1 = 1 μmol/ min/mg. Slight differences were noted for the dissociation constant ( K ia) of MgATP from the binary enzyme complex, E · MgATP. The values were 0.75 and 0.29 m m in the absence and presence of respiration. However, a 10-fold decrease in the steady-state dissociation constant ( K a) of MgATP from the ternary complex, E · MgATP · creatine, was documented: 0.15 m m with exogenous ATP and 0.014 m m with oxidative phosphorylation. Since K ia × K b does not equal K a × K ib under respiring conditions, the enzyme appears to be altered from its normal rapid-equilibrium random binding kinetics to some other mechanism by its coupling to oxidative phosphorylation.

Control of heart mitochondrial oxygen consumption by creatine kinase: the importance of enzyme localization.

The route of movement of ADP produced in the mitochondrial creatine kinase reaction was investigated by recording the rate of ADP-dependent oxygen consumption in the presence of phosphoenolpyruvate and pyruvate kinase. This pyruvate kinase system completely abolished activation of respiration by ADP added or by ADP produced in the hexokinase reaction in the medium, but was not able to inhibit the creatine kinase activated respiration when creatine kinase was bound to the inner mitochondrial membrane. These different responses of oxidative phosphorylation were observed at equal ATPADP ratios in the medium. The data obtained evidence direct channeling of ADP from heart mitochondrial creatine kinase to the adenine nucleotide translocase without its prompt release into the medium.

Heart mitochondrial creatine kinase solubilization: Effect of mitochondrial swelling and SH group reagents

The influence of mitochondrial swelling on the binding of creatine kinase to the inner membrane of pig heart mitochondria has been studied. Creatine kinase is not solubilized in isotonic mediums such as 250 m m sucrose or 125 m m KCl which do not induce mitochondrial swelling; however, it is solubilized when mitochondria swell in 100 m m potassium phosphate. This latter effect is suppressed when swelling is prevented by respiratory inhibitors or mersalyl. In hypotonic mediums creatine kinase is strongly released only when permeant anions are present; despite a high degree of swelling creatine kinase dissociation is minimal in 25 m m sucrose or KCl. Solubilization may also be obtained when swelling is induced by P j, accumulation inside mitochondria or when, after an osmotic shock in distilled water, they are resuspended in potassium phosphate or chloride but not in sucrose. Creatine kinase may be strongly released in isotonic KCl but not in sucrose when mitochondria are treated with parahydroxymercuribenzoic acid which induces swelling in the former medium (by allowing Cl − entry). A similar effect was obtained with other organic mercurials (parachloromercuriphenyl sulfonic acid, mersalyl) but not with disulfides (5,5′-dithiobis(2-nitrobenzoic acid), 6,6′-dithiodinicotinic acid, 2,2′-dithiodipyridine). Mercurials are able to solubilize creatine kinase from mitochondria suspended in distilled water so it is apparent that they have a direct effect on the association of the enzyme with the mitochondrial inner membrane. These results show that mitochondrial swelling is a prerequisite but not a sufficient condition for creatine kinase dissociation: the presence of ions or mercurials seems necessary for an efficient creatine kinase Solubilization.

Characterization of a brain particulate bound form of creatine kinase

A bound form of creatine kinase associated with brain particulate was characterized by isoelectric focusing, antigenicity and chromatography and compared to muscle (MM), brain (BB), and heart mitochondrial isoenzymes. On partial purification and isoelectric focusing, the solubilized enzyme has a pl of 7.3, similar to the pl of muscle creatine kinase MM, pl 6.8, but different from brain creatine kinase BB, which precipitates on isoelectric focusing in sucrose or glycerol stabilized media at its calculated pl of 5.6. Gel filtration chromatography of deoxycholate solubilized particulate creatine kinase on Sephadex Gl50 reveals an estimated molecular weight of approximately 80,000 daltons. The brain particulate enzyme is antigenically distinct from both muscle and rat heart mitochondrial creatine kinase isoenzymes but has antigenic similarity with soluble cytoplasmic brain BB. The situation may be analogous to that found with rat heart mitochondria and rat heart cytoplasmic isoenzymes which we have shown to exhibit antigenic similarity even though differences in electrophoretic and amino acid composition have been demonstrated; however, the confident determination that the particulate enzyme is a separate isoenzyme will have to await amino acid analysis.

The loss of heart mitochondrial creatine kinase activity: An index of functional damage: J. Bittl and W.E. Javobus Div. Cardiology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

The loss of heart mitochondrial creatine kinase activity: An index of functional damage: J. Bittl and W.E. Javobus Div. Cardiology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

Interaction between the creatine kinase of heart mitochondria and oxidative phosphorylation

Respiration dependent phosphocreatine (CP) synthesis by isolated heart mitochondria was studied under conditions designed to approximate the intracellular milieu. Oxidative phosphorylation and CP synthesis were shown to be in dynamic equilibrium, with the rate of each process being partially determined by the quasi-steady state ATP/ADP ratio of the suspending medium. Mitochondrial oxygen consumption was shown to be a function of extramitochondrial ATP/ADP ratios rather than ATP 4− /ADP 3− or ADP concentration per se when inorganic phosphate (P 1 ) was held constant. The rate of oxidative phosphorylation was also a function of extramitochondrial P 1 but not of phosphorylation potential since very high ATP/ADP ratios inhibited respiration regardless of phosphate concentration. The rate of CP synthesis was directly determined by the degree of displacement of the creatine kinase (CPK) mass action quotient from equilibrium, and the observed respiration dependent stimulation of CP synthesis from ATP and creatine appeared related to the high extramitochondrial ATP/ADP ratios observed during oxidative phosphorylation. When similar ATP/ADP ratios were maintained by adding pyruvate kinase and phosphoenolpyruvate (PEP) to non-respiring mitochondria, the rate of CP synthesis was comparable to that observed during respiration. Labelling studies indicated that extramitochondrial ATP is the source of CP phosphate, and no privileged access of matrix ATP for mitochondrial CPK could be detected. These studies suggest that the myocardial creatine kinase system promotes rapid equilibration of CPK substrates throughout the cytoplasmic compartment. Accordingly, fluctuations of ATP and ADP are minimized while CP/creatine ratios and P 1 communicate energy demands of the contractile system to the mitochondria.

Creatine Kinase of Rat Heart Mitochondria: COUPLING OF CREATINE PHOSPHORYLATION TO ELECTRON TRANSPORT

Abstract Intact mitochondria from human, beef, and rat heart, as well as rat brain and rat skeletal and smooth muscle, contain considerable creatine kinase activity; those from rat or rabbit liver, kidney, and testis contain essentially none. In rat heart mitochondria creatine kinase activity is far higher than the activity of adenylate kinase and nucleoside diphosphokinase. The heart mitochondrial creatine kinase is specific for ATP and ADP as phosphate donor and acceptor. The optimum pH is 6.7 in the direction of ATP formation and 8.0 in the direction of phosphocreatine formation. The mitochondrial isoenzyme of creatine kinase is not present in the same compartment as the intermembrane marker enzyme adenylate kinase, but may be released in soluble form by exposure to sodium acetate or sodium phosphate medium at pH 7.4. The phosphorylation of creatine by intact mitochondria takes place outside the atractyloside-sensitive ADP-ATP carrier system of the inner membrane and thus requires external ATP. When creatine in the concentration range known to be present in intact heart is added to rat heart mitochondria neither the initial State 4 or State 3 rate of respiration (succinate) is affected; however, there is a large increase in the State 4 rate following addition of ADP, whose magnitude increases as the creatine concentration is increased. In the presence of physiological concentrations of creatine and ADP rat heart mitochondria respire at a constant high rate equal to 75 to 85% of their maximal State 3 rate; under these conditions phosphocreatine is the end product of oxidative phosphorylation. The Km values for rat heart mitochondria are 35 µm for ADP, 0.72 mm for phosphocreatine, 100 µm for ATP, and 6 mm for creatine. These and other properties suggest that in addition to the ∼P storage function usually postulated for it, the phosphocreatine-creatine kinase system serves to smooth out or buffer the rate of respiration of heart mitochondria at a high and constant rate, as well as to channel ∼P to the contractile system via the mitochondrial and sarcoplasmic isoenzymes of creatine kinase.