The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

Physiological role of the creatine kinase system and the problem of regulating the activity of mitochondrial creatine kinase

The review contains the analysis of present-day concepts on the physiological role of the creatine kinase system. A hypothesis on the buffering functions of the creatine kinase system which ensures a constant ATP level in cells and a hypothesis according to which phosphocreatine is a macroergic phosphate carrier from mitochondria to the sites of their utilization are considered. In connection with the creatine phosphate carrier hypothesis according to which the transport function of the creatine kinase system is provided for by an effective function of mitochondrial creatine kinase, feasible mechanisms of mitochondrial creatine kinase activity regulation are considered: as a result of creation of local concentration of nucleotide substrates as well as changes in the properties of creatine kinase itself which may result from the enzyme conversion from the membrane-bound to the free form or from the interconversion of oligomeric forms of the enzyme.     

Alterations in the creatine kinase system in the myocardium of cardiomyopathic hamsters

Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.     

Purification and characterization of human mitochondrial creatine kinase. A single enzyme form

Purification of human mitochondrial creatine kinase has been difficult and procedures that were highly successful in purifying canine enzyme failed for human mitochondrial creatine kinase. In the present study, we employed ultracentrifugation to remove the lipid, urea to prevent aggregation, followed by a final step of chromatofocusing which yielded a preparation of human mitochondrial creatine kinase with a specific enzyme activity of greater than 400 IU/mg. Biochemical and immunological characterization showed the preparation to be highly pure and free of even trace amounts of other creatine kinase isoenzymes. Antiserum specific for mitochondrial creatine kinase was developed which exhibited no cross-reactivity to cytosolic creatine kinase and mitochondrial creatine kinase did not cross-react with antiserum to the cytosolic forms. Marked differences were noted, both biochemically and immunologically, between mitochondrial creatine kinase and the cytosolic forms. Human mitochondrial creatine kinase was shown to have a molecular weight of around 82,000 and to be composed of two subunits of equal molecular weights around 41,000. Aggregates of mitochondrial creatine kinase were observed with molecular weights of around 200,000 in the absence of urea or if isolated from material after having undergone proteolysis. Isolation from fresh material or in the presence of urea inhibited aggregate formation for both canine and human mitochondrial creatine kinase. Despite claims of several investigators that mitochondrial creatine kinase exhibits two to three forms with varying molecular weights, our data indicate a single enzyme form made up of a subunit with a molecular weight of 41,000 and the high molecular weight aggregates appear to be induced artifacts. A radioimmunoassay was developed for human mitochondrial creatine kinase which, with appropriate modifications, should detect mitochondrial creatine kinase in human plasma.     

Inactivation of rabbit muscle creatine kinase by reversible formation of an internal disulfide bond induced by the fungal toxin gliotoxin

The biological activity of gliotoxin is dependent on the presence of a strained disulfide bond that can react with accessible cysteine residues on proteins. Rabbit muscle creatine kinase contains 4 cysteines per 42-kDa subunit and is active in solution as a dimer. Only Cys-282 has been identified as essential for activity. Modification of this residue results in loss of activity of the enzyme. Treatment of creatine kinase with gliotoxin resulted in a time-dependent loss of activity abrogated in the presence of reducing agents. Activity was restored when the inactivated enzyme was treated with reducing agents. Inactivation of creatine kinase by gliotoxin was accompanied by the formation of a 37-kDa form of the enzyme. This oxidized form of creatine kinase was rapidly reconverted to the 42-kDa species by the addition of reducing agents concomitant with restoration of activity. A 1:1 mixture of the oxidized and reduced monomer forms of creatine kinase as shown on polyacrylamide gel electrophoresis was equivalent to the activity of the fully reduced form of the enzyme consistent with only one reduced monomer of the dimer necessary for complete activity. Conversion of the second monomeric species of the dimer to the oxidized form by gliotoxin correlated with loss of activity. Our data are consistent with gliotoxin inducing the formation of an internal disulfide bond in creatine kinase by initially binding and possibly activating a cysteine residue on the protein, followed by reaction with a second neighboring thiol. The recently published crystal structure of creatine kinase suggests the disulfide is formed between Cys-282 and Cys-73.     

The role of the mitochondrial creatine kinase system for myocardial function during ischemia and reperfusion.

The subcellular distribution of ATP, ADP, creatine phosphate and creatine was studied in normoxic control, isoprenaline-stimulated and potassium-arrested guinea-pig hearts as well as during ischemia and after reperfusion. The mitochondrial creatine phosphate/creatine ratio was closely correlated to the oxidative activity of the hearts. This was interpreted as an indication of a close coupling of mitochondrial creatine kinase to oxidative phosphorylation. To further investigate the functional coupling of mitochondrial creatine kinase to oxidative phosphorylation, rat or guinea-pig heart mitochondria were isolated and the mass action ratio of creatine kinase determined at active or inhibited oxidative phosphorylation or in the presence of high phosphate, conditions which are known to change the functional state of the mitochondrial enzyme. At active oxidative phosphorylation the mass action ratio was one-third of the equilibrium value whereas at inhibited oxidative phosphorylation (N2, oligomycin, carboxyatractyloside) or in the presence of high phosphate, the mass action ratio reached equilibrium values. These findings show that oxidative phosphorylation is essential for the regulation of the functional state of mitochondrial creatine kinase. The functional coupling of the mitochondrial creatine kinase and oxidative phosphorylation indicated from the correlation of mitochondrial creatine phosphate/creatine ratios with the oxidative activity of the heart in situ as well as from the deviation of the mass action ratio of the mitochondrial enzyme from creatine kinase equilibrium at active oxidative phosphorylation in isolated mitochondria is in accordance with the proposed operation of a creatine shuttle in heart tissue.     

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.     

Decreased Creatine Kinase Activity Caused by Electroconvulsive Shock

Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy to minimize side effects of this treatment, little progress has been made in understanding its mechanism of action. Creatine kinase is a crucial enzyme for brain energy homeostasis, and a decrease of its activity has been associated with neuronal death. This work was performed in order to evaluate creatine kinase activity from rat brain after acute and chronic electroconvulsive shock. Results showed an inhibition of creatine kinase activity in hippocampus, striatum and cortex, after acute and chronic electroconvulsive shock. Our findings demonstrated that creatine kinase activity is altered by electroconvulsive shock.     

Adenosine Diphosphate Requirement in the Creatine Phosphate-Induced Contraction of the Glycerinated Rabbit Psoas

Glycerinated rabbit psoas fibers were tested for their ability to contract under the influence of creatine phosphate and creatine kinase in the absence of free nucleotide. Tension development by the fibers was observed upon addition of creatine phosphate to the medium containing creatine kinase purified to the first lyophilization stage. However, when the enzyme was washed free of nucleotides by treatment with the anion exchange resin Dowex 1, no contraction occurred until free nucleotide was supplied. In all experiments, contractile activity of the psoas fibers was the criterion for determining the enzyme activity concerned. Using this criterion, creatine kinase activity native to the glycerinated fibers was also demonstrated. No evidence for direct transphosphorylation of the bound nucleotide of the fiber was found.     

Inactivation of creatine kinase by high pressure may precede dimer dissociation.

The effects of hydrostatic pressure on creatine kinase activity and conformation were investigated using either the high-pressure stopped-flow method in the pressure range 0.1-200 MPa for the activity determination, or the conventional activity measurement and fluorescence spectroscopy up to 650 MPa. The changes in creatine kinase activity and intrinsic fluorescence show a total or partial reversibility after releasing pressure, depending on both the initial value of the high pressure applied and on the presence or absence of guanidine hydrochloride. The study on 8-anilinonaphthalene-1-sulfonate binding to creatine kinase under high pressure indicates that the hydrophobic core of creatine kinase was progressively exposed to the solvent at pressures above 300 MPa. This data shows that creatine kinase is inactivated at low pressure, preceding both the enzyme dissociation and the unfolding of the hydrophobic core occurring at higher pressure. Moreover, in agreement with the recently published structure of the dimer, it can be postulated that the multistate transitions of creatine kinase induced both by pressure and guanidine denaturation are in direct relationship with the existence of hydrogen bonds which maintain the dimeric structure of the enzyme.     

Modification of contractile proteins by oxygen free radicals in rat heart

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effect of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time-and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was protected by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.     

Rate equation for creatine kinase predicts the in vivo reaction velocity: 31P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat

Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 +/- 2.8, 62.4 +/- 4.5, and 224 +/- 16 mM/s) and exceeded the NMR-determined in vivo reaction velocities by an order of magnitude (4.1 +/- 1.2, 5.1 +/- 1.6, and 18.4 +/- 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues: greater than 99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98% MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p less than 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.     

Phosphagen kinase evolution. Expression in echinoderms

Arginine kinase and creatine kinase that catalyze the transfer of a phosphate group between ATP and arginine and creatine, respectively, play an important role in cellular energetics. In contrast to most animals which exhibit a single phosphagen kinase activity (creatine kinase in chordates and arginine kinase in protostomians), echinoderms exhibit both arginine kinase and creatine kinase activities, sometimes in the same tissue. In contrast to chordates in which creatine kinases are dimers (consisting of two subunits of 40 kDa) and protostomians in which arginine kinases are usually monomers (40 kDa), echinoids contain specific phosphagen kinases: a dimeric arginine kinase (consisting of two subunits of 42 kDa) in eggs and a monomeric creatine kinase (145 kDa) in sperm. We have examined echinoderms from the five existing classes (echinoids, asteroids, ophiuroids, holothurians and crinoids) for the expression of these specific phosphagen kinases in different tissues. Gel filtration was used to determine the molecular masses of the native enzymes. Antibodies specific for arginine kinase or for creatine kinase were used to characterize the subunit composition of arginine kinase and creatine kinase after SDS/PAGE and transfer. In all echinoderms analyzed, arginine kinase always occurred as an enzyme of about 81 kDa consisting of two subunits of 42 kDa and creatine kinase as a monomeric enzyme of 140-155 kDa. The occurrence in echinoderms of both phosphagen kinases with distinct specificities and specific molecular structures is discussed from both a developmental and evolutionary point of view.     

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.     

Cytosolic adenylate kinase catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate

An attempt was made to purify a porcine skeletal muscle enzyme catalyzing the formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP), requiring ATP, Mg2+ and a cofactor (creatine). As the purification proceeded, the reaction requirements for ATP and creatine were lost and then a requirement for ADP was manifested. The activity responsible for TTP synthesis from TDP, ADP, and Mg2+ was found to be copurified with adenylate kinase [EC 2.7.4.3] activity, and was finally purified to a single band on SDS-PAGE. Antiserum obtained against the purified enzyme preparation inhibited both adenylate kinase activity and the TTP-synthesizing activity to exactly the same extent. These results indicate that adenylate kinase catalyzes TTP formation from TDP in vitro.     

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.     

Identification of creatine as a cofactor of thiamin-diphosphate kinase

Thiamin-diphosphate (TDP) kinase which catalyzes thiamin triphosphate formation from TDP requires a low-molecular-mass cofactor in addition to ATP and Mg2+. The cofactor was isolated in a crystalline form from pig skeletal muscle and identified as creatine by proton NMR, mass spectrometry, infrared spectrometry and elemental analysis. The isolated cofactor and authentic creatine supported the same activity of partially purified TDP kinase at identical molar concentrations. Neither creatine phosphate nor creatinine showed activity as a cofactor. This is the first report showing evidence of the existence of a creatine-dependent enzyme.     

Creatine induction of creatine kinase synthesis in a developing monolayer culture of muscle cells]

Creatine action on the activity of creatine kinase (ATP: creatine-phosphotransferase; EC 2.7.3.2) and the content of water-soluble proteins in the developing monolayer culture of chick myoblasts are studied. Creatine at concentrations of 1.9-10- minus 3-3.8-10- minus 3 M is shown to increase reliably the creatine kinase activity by 1,1--2,9 times and to reduct considerably the content of water-soluble proteins. Lower concentrations of creatine (3.8-10- minus 5 M) also increased the creatine kinase activity but did not change the contents of water-soluble proteins. The creatine effect was maximal at the period preceding the termination of tissue cells differentiation. In the course of the combined effect of both actinomycin D (50 mcg/plate) and creatine (3.8-10- minus 3 M) the creatine kinase activity was much higher than that in the presence of actinomycin D alone which considerably reduced the enzyme activity as well as the contents of water-soluble proteins.     

Aggregation of creatine kinase during refolding and chaperonin-mediated folding of creatine kinase

The course of refolding and reactivation of urea-denatured creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) has been studied in the absence and presence of molecular chaperonin GroEL. The enzyme was denatured in Tris--HCl buffer containing 6 M urea for 1 h. In the refolding studies, the denatured enzyme was diluted 60-fold into the same buffer containing GroEL or not for activity, turbidity, fluorescence measurements and polyacrylamide gel electrophoresis. The results show that the reactivation process is dependent of creatine kinase concentration in the concentration range 2.5--4 microM. The levels of activity recovery decrease with increasing enzyme concentration because of the formation of wrong aggregates. The molecular chaperonin GroEL can bind the refolding intermediate of creatine kinase and thus prevent the formation of wrong aggregates. This intermediate is an inactive dimeric form that is in a conformation resembling the 'molten globule' state.     

Contact site between inner and outer mitochondrial membrane: a dynamic microcompartment for creatine kinase activity

Creatine kinase is involved in the integration of high-energy metabolism in various tissues. In this study the tissue-specific distribution of the mitochondrial isoform was investigated, both by electrophoresis of rat tissue extracts, and by ultrastructural localisation of creatine kinase activity. Furthermore, the influence of uncoupling of oxidative phosphorylation on mitochondrial creatine kinase activity associated with intermembrane contacts was investigated by enzyme cytochemistry and morphometric analysis. The results of the cytochemical survey indicate that contact sites are a prerequisite for creatine kinase to demonstrate enzymatic activity. Moreover, the extent of creatine kinase active membrane contacts depends on the metabolic state of the mitochondrion, as shown for heart mitochondria in vivo and in vitro, before and after treatment with dinitrophenol.