Serum creatine kinase isoenzymes in progressive muscular dystrophy

Creatine kinase isoenzymes in sera and muscle biopsies obtained from 50 controls, 72 patients with progressive muscular dystrophy (PMD), 68 patients with other neuromuscular disorders, 17 carriers of Duchenne-type PMD and 15 patients with myocardial infarction were studied. MB isoenzyme was detected in the sera of 58 patients with PMD and 56 out of 61 muscle biopsies. The MB activity varied between 4 and 400 IU/1 or 3.4--22% of total activity. The MB activity was demonstrated in a considerably smaller number of cases with polymyositis, dystrophic myotonia and Kugelberg-Welander disease. The MB isoenzyme in sera of PMD persisted for many years. It is admitted that the MB isoenzyme in the serum of patients with PMD originates chiefly from skeletal muscle.     

Serum creatine kinase activity following forearm flexion isometric exercise

The purpose of this study was to compare serum creatine kinase (CK) activity following two forearm flexion isometric exercise regimens differing in work to rest ratio, and examine the CK response to a repeated bout of isometric exercise. Eleven males were tested on two sessions (bouts) spaced 1 week apart. For bout 1, five subjects (group A) performed a forearm flexion isometric exercise consisting of 40 10-s maximal contractions with 20-s inter-trial rests (10:20), while six (group B) performed 40 maximal 10-s contractions with 5-s inter-trial rests (10:5). The increase in serum CK activity following the 10:20 exercise (143%) was significantly greater than that following the 10:5 exercise (52%). The 10:20 exercise was also associated with greater tension generation over trials. One week later, both groups performed a bout of 10:20 exercise. A substantial reduction in the serum CK response was found following this second bout. The data suggest that for bout 1 the isometric exercise associated with the greater overall tension levels resulted in the greater CK response. However, when the 10:20 exercise was repeated 1 week later, a substantial reduction in the CK response was found which was unrelated to the tension generated.     

Serum creatine phosphokinase levels in rats following cold injury

The validity of serum creatine phosphokinase (CPK) as an index of tissue damage due to cold injury was tested in the rat. Immersion of the feet in the freezing mixture (salt and ice, –19°±1°C) for one or five minutes produced reversible and irreversible damage, respectively. The increase in CPK activity over a 5-hr period following freezing showed that the severity and extent of cold injury was associated with the amount of CPK released into the bloodstream. -Exposure of the whole rat to cold air at –29°C for three hours resulted in a highly variable increase in serum CPK two hours after exposure which was related to the degree of hypothermia during the exposure. The extent of hypothermia was also related to the amount of tissue loss estimated two weeks after the exposure. These findings show that the increase in serum CPK after cold injury reflects subsequent tissue loss and might conceivably be useful in early classification of cold injury.

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Zusammenfassung Die Verwendbarkeit der Serumkreatinphosphatase (CPK) als Indikator von Gewebsschäden nach Erfrierungen wurde an der Ratte untersucht. Eintauchen der Pfote in eine Kältemischung (Salz und Eis, –19°±1°C) über Perioden von 1–5 Minuten führte zu reversiblen und irreversiblen Gewebsschädigungen. Der Anstieg der CPK-Aktivität 5 Stunden nach Beginn der Erfrierungen zeigte, dass die Schwere und das Ausmass des Kälteschadens im Zusammenhang stand mit der in das Blut ausgetretenen CPK-Menge. - Aussetzen von Ratten während 3 Stunden bei –29°C führte zu einem Anstieg der Serum CPK 2 Stunden später. Die Werte wiesen eine grosse Streuung auf, die im Zusammenhang stand mit dem Ausmass der Hypothermie während der Belastung. Die Hypothermie war positiv korreliert mit der Menge des Gewebsverlustes 2 Wochen später. Danach ist der Anstieg der Serum-CPK nach Erfrierungen Ausdruck der Gewebsschädigung und kann eventuell zur frühen Beurteilung von Erfrierungen dienen.

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Resume Des rats ont servi de bêtes d'essai pour vérifier si l'analyse de la phosphatase de la créatine du sérum (CPK) est utilisable comme indicateur de dommages des tissus après des phénomènes de gelures. Le fait de placer les pattes des rats dans un mélange réfrigérant (mélange de glace et de sel; température –19° à ± 1°C) durant des périodes de 1 à 5 minutes a conduit à des dégâts réversibles et irréversibles des tissus. L'augmentation de l'activité de CPK cinq heures après le début des gelures a montré que l'importance des lésions était en liaison directe avec les quantités de CPK présentes dans le sang. L'exposition de rats durant 3 heures à une température de –29°C a conduit à une augmentation du taux de CPK du sérum deux heures après. Les valeurs recueillies présentent cependant une très grande variabilité qui est en relation directe avec l'importance de l'hypothermie durant la période de contrainte. Cette hypothermie est en corrélation positive avec la quantité de tissu perdu deux semaines après l'exposition. Il ressort de cette expérience que l'augmentation de CPK dans le sérum sanguin après des gelures peut être utilisée comme expression des dégâts subis par les tissus et éventuellement pour la détermination précoce de phénomènes de gelures.

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DCIEM Research Paper 913     

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency. This demonstrates that the gamma-phosphate and the 2' and 3' hydroxyls are not involved in the covalent linkage and that all three phosphates, the ribose and base of the ATP molecule are retained upon autoincorporation (nucleotidylation). Treatment with NaIO3 to break the 2'-3' linkage effected total loss of radiolabel indicating that nucleotidylation resulted in opening of the ribose ring at the C1' position. Nucleotidylation with increasing [alpha32P]ATP at 37 degrees C gives an approximate k0.5 of 125 microM and saturates at 340 microM nucleotide. Modification of 8-10% of the copy numbers occurs at saturation, and CK activity is inhibited to approximately the same degree. Low micromolar levels of native substrates such as ADP, ATP, and phosphocreatine substantially reduce [alpha32P]ATP nucleotidylation. In contrast, AMP, GTP, GMP, NADH, and creatine did not effectively reduce nucleotidylation. When [alpha32P]ATP-nucleotidylated or [alpha32P]8N3ATP-photolabeled CK is treated with trypsin a single, identical radiolabeled peptide (V279-R291) is generated that comigrates on reverse phase HPLC and Tris-tricine electrophoresis. Nucleotidylation into this peptide was prevented 86% by the presence of ATP. We conclude that CK is nucleotidylated within the active site by modification at the C1'position and that autophosphorylation of this enzyme does not occur.     

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.     

The role of creatine kinase and arginine kinase in muscle.

Arginine and creatine kinase activities in different muscles are compared with calculated maximum rates of ATP turnover. The magnitude of the kinase activities decreases in the following order: anaerobic muscles and vertebrate skeletal muscles greater than heart muscle greater than insect flight muscle. The maximum activity of phosphagen kinases (i.e. creatine kinase and arginine kinase), in the direction of phosphagen formation, is lower than the calculated maximum rate of ATP turnover in insect flight muscle or rat heart.     

Serum creatine kinase activity following repeated bouts of isometric exercise with different muscle groups

The purpose of this study was to examine the relationship between the muscle mass involved in exercise and post-exercise serum creatine kinase (CK) elevation. Twelve untrained college-aged men completed three isometric exercises: one arm flexion (OAF), two arm flexion (TAF) and one leg knee extension (OLE). These exercises were balanced over subjects and days and separated by two week intervals. Each exercise consisted of 40 maximal isometric concentrations lasting for 10 s with a 20 s rest between contractions. Relative increases in serum CK for OAF, TAF, and OLE were 181 +/- 70% (SD), 222 +/- 69% and 297 +/- 67%, respectively. An ANOVA using a latin square design for analysis of carry over effects showed that these CK increases were not significantly different (p greater than 0.05). However, the increase in serum CK following the first exercise (379 +/- 90%), regardless of what it was (OAF, TAF, or OLE), was significantly greater (p less than 0.05) than those following bouts 2 and 3 (155 +/- 29%; 167 +/- 54%). Regression analysis indicated that post-exercise serum CK elevation was not related to the amount of muscle mass involved in the exercise (r = 0.30, p greater than 0.05) nor to muscle tension developed (r = 0.28, p greater than 0.05). We conclude that post-exercise serum CK elevation is not necessarily related to the muscle mass involved in the exercise. Because each exercise involved the use of different muscle groups, factors outside the exercising muscle may contribute to post-exercise serum enzyme activity.     

Serum creatine kinase activity varies with ovulatory status in regularly exercising, premenopausal women

BACKGROUND/AIMS: The clinical complications associated with an unopposed estrogen environment and luteal phase defects observed in exercising women prompted the examination of the relationship of exercise and endogenous ovarian steroids with serum creatine kinase (CK) activity. METHODS: Subjects (n = 34) were classified into three groups according to their exercise and menstrual status, sedentary and exercising ovulatory groups (SedOvul, ExOvul), and an exercising amenorrheic group (ExAmen). Daily urine samples were collected to assess urinary ovarian steroid exposure and menstrual status. Serum CK activity was assayed in each menstrual cycle of all subjects. RESULTS: Exercise increased serum CK activity in all exercising subjects (p < 0.01), but the increase was greater in amenorrheic women compared to ovulatory women (SedOvul: 33.0 +/- 3.4; ExOvul: 43.7 +/- 4.1; ExAmen: 54.4 +/- 3.6, p < 0.05). When the ovulatory women were further divided into those with normal steroid production (ExOvul subgroup) and those with a suppressed progesterone luteal phase environment (ExLPD), both the ExOvul (51.9 +/- 5.4 IU/l) subgroup and ExAmen group had higher serum CK activity (p < 0.05) than the ExLPD (36.6 +/- 5.2 IU/l) subjects or the sedentary controls. CONCLUSIONS: These data demonstrate the complex association between ovarian hormone status and the normal serum CK response to regular mechanical stress imposed by chronic exercise training.     

The creatine kinase system and pleiotropic effects of creatine

The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.     

Mouse p53 represses the rat brain creatine kinase gene but activates the rat muscle creatine kinase gene.

The creatine kinases (CK) regenerate ATP for cellular reactions with a high energy expenditure. While muscle CK (CKM) is expressed almost exclusively in adult skeletal and cardiac muscle, brain CK (CKB) expression is more widespread and is highest in brain glial cells. CKB expression is also high in human lung tumor cells, many of which contain mutations in p53 alleles. We have recently detected high levels of CKB mRNA in HeLa cells and, in this study, have tested whether this may be due to the extremely low amounts of p53 protein present in HeLa cells. Transient transfection experiments showed that wild-type mouse p53 severely repressed the rat CKB promoter in HeLa but not CV-1 monkey kidney cells, suggesting that, in HeLa but not CV-1 cells, p53 either associates with a required corepressor or undergoes a posttranslational modification necessary for CKB repression. Conversely, mouse wild-type p53 strongly activated the rat CKM promoter in CV-1 cells but not in HeLa cells, suggesting that, in CV-1 cells, p53 may associate with a required coactivator or is modified in a manner necessary for CKM activation. The DNA sequences required for p53-mediated modulations were found to be within bp -195 to +5 of the CKB promoter and within bp -168 to -97 of the CKM promoter. Moreover, a 112-bp fragment from the proximal rat CKM promoter (bp -168 to -57), which contained five degenerate p53-binding elements, was capable of conferring p53-mediated activation on a heterologous promoter in CV-1 cells. Also, this novel p53 sequence, when situated in the native 168-bp rat CKM promoter, conferred p53-mediated activation equal to or greater than that of the originally characterized far-upstream (bp -3160) mouse CKM p53 element. Therefore, CKB and CKM may be among the few cellular genes which could be targets of p53 in vivo. In addition, we analyzed a series of missense mutants with alterations in conserved region II of p53. Mutations affected p53 transrepression and transactivation activities differently, indicating that these activities in p53 are separable. The ability of p53 mutants to transactivate correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a and activated Ras.     

Serum and organ creatine phosphokinase alterations in exercise.

Rats that swam for 3 h showed a 6-fold increase in serum creatine phosphokinase (SCPK) activity which declined to control values within 7 h after swimming. Of the excess SCPK, 77% was BB isoenzyme; the remainder was mainly MM with traces of MB. Kidney, liver, brain and lung contain mainly BB (50-80%) and only a trace of MB (0-7%). Heart CPK was composed of little BB (8%) and more MB (28%) and MM (64%). Skeletal muscle CPK was almost entirely MM. CPK activity is highest in skeletal muscle, intermediate in heart and brain and lowest in kidney, liver and lung. It is suggested that skeletal muscle and heart are not involved in CPK release in swimming, and kidney, liver and brain may be sites of release.     

Two-state irreversible thermal denaturation of muscle creatine kinase.

Thermal denaturation of creatine kinase from rabbit skeletal muscle has been studied by differential scanning calorimetry. The excess heat capacity vs. temperature profiles were independent of protein concentration, but strongly temperature scanning rate-dependent. It has been shown that thermal denaturation of creatine kinase satisfies the previously proposed validity criteria for the two-state irreversible model [Kurganov et al., Biophys. Chem.70 (1997) 125]. The energy activation value has been calculated to be 461.0 +/- 0.7 kJ/mol.     

Structural changes of creatine kinase upon substrate binding.

Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.     

Phenylbutazone radicals inactivate creatine kinase

Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by phenylbutazone (PB), an effective drug to treat rheumatic and arthritic diseases, with horseradish peroxidase and hydrogen peroxide (HRP-H₂O₂). PB inactivated CK during its interaction with HRP-H₂O₂, and inactivated CK in rat heart homogenate. PB carbon-centered radicals were formed during the interaction of PB with HRP-H₂O₂. The CK efficiently reduced electron spin responance signals of the PB carbon-centered radicals. The spin trap agent 2-methyl-2-nitrosopropane strongly prevented CK inactivation. These results show that CK was inactivated through interaction with PB carbon-centered radicals. Sulfhydryl groups and tryptophan residues in CK were lost during the interaction of PB with HRP-H₂O₂, suggesting that cysteine and tryptophan residues are oxidized by PB carbon-centered radicals. Other enzymes, including alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, but not lactate dehydrogenase, were also inactivated. Sulfhydryl enzymes seem to be sensitive to attack by PB carbon-centered radicals. Inhibition of SH enzymes may explain some of the deleterious effects induced by PB.