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At least 163 records · Page 9Linked to original sources
PubMed1989

At what level of serum total creatine kinase activity can measurement of serum creatine kinase MB isoenzyme activity be omitted in suspected myocardial infarction?

The purpose of this study was to establish a discriminatory limit for serum total creatine kinase activity (CK activity) below which CK isoenzyme fractionation is unnecessary. We looked at 2610 serum samples from 1077 consecutive patients with suspected acute myocardial infraction (AMI). The CK activity was determined according to the Scandinavian recommended method. Isoenzymes of CK were separated by agarose gel electrophoresis, followed by fluorometric scanning. When the threshold for CK activity was 150 U/l, none of the samples had a creatine kinase MB isoenzyme activity (CK-MB activity) equal to or higher than 30 U/l (the diagnostic level), which has been found to differentiate between patients with AMI and those without AMI. Only 14 patients (1.3% of all patients investigated) had CK-MB activity peaks between 10 U/l (detection limit) and 30 U/l. Of these, AMI was only diagnosed in one. We recommend that CK-MB activity should be measured only when CK activity is higher than 150 U/l. This would make about 50% of all CK-MB measurements unnecessary.

Aged↗
PubMed1976

Evaluation of adenosine 5'-monophosphate and fluoride as adenylate kinase inhibitors in the creatine kinase assay.

Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase.

Adenosine Monophosphate↗
PubMedDate not supplied

[Creatine kinase activity and isoenzymic spectrum of myocardium creatine kinase in rats of different age].

In old rats the specific activity of creatine kinase (EC 2.7.32) in homogenate, mitochondrial and postmitochondrial fractions of the myocardium is essentially lower than in adult animals. It does not change with age in myofibrils. The isoenzymic spectrum of creatine kinase is unchanged as well. In old rats the content of creatine phosphate in the myocardium is considerably lower, and that of creatine is higher than in young animals. This evidences for a decrease in the rate of creatine phosphorylation controlled by creatine kinase.

Aging↗
PubMed1983

Tetrathionate-blocked creatine kinase as a substrate for human plasma 'creatine kinase conversion factor'.

'Creatine kinase conversion factor' has been partially purified from whole plasma. The preparation has the ability to convert slowly migrating, native CK-MM into a faster electrophoretic form through a form of intermediate mobility. These changes are accompanied by a loss in CK activity. The rates of both electrophoretic conversion and loss of enzyme activity are reduced or prevented by the presence of 2-mercaptoethanol, EDTA, or by formation of the creatine-MgADP-nitrate dead-end complex. When the thiol group essential for enzyme activity found at the CK active site is first blocked by treatment with potassium tetrathionate, full activity may be retained under conditions that fully convert CK to the faster electrophoretic forms. The conversion factor activity was not inhibited by a pre-incubation with the protease inhibitor, PMSF and the converted form of CK showed no evidence of proteolytic cleavage. The molecular basis of the chemical alteration caused by the conversion is discussed.

Creatine Kinase↗
PubMed1994

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.

Animals↗
PubMed1990

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.

Animals↗
PubMed1991

Creatine kinase determination: a European evaluation of the creatine kinase determination in serum, plasma and whole blood with the Reflotron system.

We evaluated a new dry-reagent carrier system for the determination of creatine kinase (EC 2.7.3.2) activity, Reflotron CK, with special attention to analytical performance with whole blood. We found a good within series imprecision. The median coefficient of variation was 3.1% for Reflotron CK (blood, serum and plasma) and 0.9% for the automatic analysers (serum and plasma only). The between-days imprecision with Reflotron CK (median CV: less than or equal to 3%) was similar to that for the comparison method on different analysers. Fresh samples of human blood, plasma and serum were examined by Reflotron CK and by a N-acetylcysteine activated creatine kinase method in six different clinical laboratories and in the Evaluation Department of Boehringer Mannheim GmbH. The correlation between these methods was excellent (r greater than or equal to 0.99), the median systematic deviation (bias) for all samples being smaller than -5%. Haematocrits between 0.25 and 0.50, haemolysis up to 6 g/l haemoglobin, and icteric samples with bilirubin concentrations up to 0.2 g/l showed no interference. No drug in therapeutic concentration was found to affect the Reflotron CK results; ascorbic acid, calcium dobesilate and sulphamethoxazole lowered the values only when present in high concentrations. Reflotron CK may be considered as a suitable alternative for decentralized testing sites, especially in situations where creatine kinase results are needed quickly.

Autoanalysis↗
PubMed1981

Creatine kinase in tibial shaft fractures. Serum creatine kinase levels in patients with tibial shaft fractures.

The creatine kinase (CK) activity of the serum of 33 male and 24 female patients with tibial shaft fractures has been assayed. In 40 of the 57 patients the CK level surpassed the maximal normal limit of 1.7 mukat/l. Patients with fractures due to direct violence had significantly higher levels than those with fractures due to indirect violence. When the fracture was displaced the CK level was more often abnormal than when there was no displacement. Patients with extensive swelling of the injured leg had significantly higher levels than patients with minor or no swelling. CK determinations could be used to quantify muscle injury.

Adolescent↗
PubMed1993

Recombinant creatine kinase proteins and proposed standards for creatine kinase isoenzyme and subform assays.

We developed standards for creatine kinase (CK; EC 2.7.3.2) assays by expressing human CK cDNAs in COS cells. Cells were transiently transfected with full-length cDNAs for CK subunits M and B, individually and in combination; and subsequently, high concentrations of CK activity were present in the cell lysate (1.2 U/mg protein). These proteins exhibited the characteristic isoenzyme-specific electrophoretic mobilities for CK MM and BB isoenzymes. We also produced subforms of CK MM and MB, identical to the modified CK variants produced in plasma after muscle or myocardial injury, by mutating the cDNA for the CK M subunit to delete the carboxy-terminal lysine residue. When this construct was cotransfected with the normal cDNAs for CK M and B, five electrophoretically distinct CK isoenzymes were detected by nondenaturing electrophoresis: MM3, MM2, MM1, MB2, and MB1. These proteins retained 100% of their activity after storage of the cell lysates -20 or 4 degrees C for 3 months.

Base Sequence↗
PubMed1992

Ratio of creatine kinase 2 mass concentration to total creatine kinase activity not altered by heavy physical exercise.

Serum creatine kinase isoenzyme 2 concentrations (CK 2 mass) were measured in marathon runners during training and 1 and 2 days after a race and compared with values from 36 acute myocardial infarction (AMI) patients whose total CK and (or) CK 2 activities were similar to those of runners in the basal state. During training, runners had CK and CK 2 activities 53% and 43% above reference values, respectively, and 36% had CK 2 activity > 5% of total CK. Nine runners (26%) showed CK 2 mass values > 6 micrograms/L but < or = 10 micrograms/L; 35 of the AMI subjects, despite having CK activities similar to those of runners, had values > 10 micrograms/L. The ratio of CK 2 mass to total CK activity was significantly (P < 0.0002) different between sexes for runners. At 1 and 2 days after racing, 100% of CK and CK 2 activities and 71% and 57% of the percentages of CK 2 activity, respectively, were abnormally high; 57% and 43% of CK 2 mass values were > 10 micrograms/L, being comparable with those observed for the AMI group. Basal CK 2 mass values of the runners appeared only slightly higher than that for sedentary subjects, but after exercise half the subjects presented increased values similar to those observed for AMI subjects. The ratio of CK 2 mass to total CK activity appeared unaltered by exercise in all but one of the samples assayed, indicating its utility in evaluating CK 2 mass increases originating in skeletal muscle.

Adult↗
PubMed1988

Plasma pyruvate kinase activity vs creatine kinase activity as an indicator of the porcine stress syndrome.

Plasma pyruvate kinase (PK) and creatine kinase (CK) activities were increased significantly (P less than 0.001 and P less than 0.05, respectively) in homozygote halothane-reacting pigs (nn), compared with those activities in homozygote nonreacting pigs (NN). Pyruvate kinase activity was less variable within groups than was CK activity, allowing more effective discrimination between nn and NN geno-types. The PK and CK activities in plasma increase with age in halothane-reacting pigs and the nonreacting pigs. Enzyme activities in heterozygote (Nn) nonreacting pigs did not differ significantly (P greater than 0.05) from enzyme activities of homozygote (NN) nonreacting pigs. Although PK was better than CK in identifying stress-susceptible pigs, age-related effects and the failure to identify heterozygotes may restrict the use of plasma PK activity as a diagnostic test for the stress syndrome.

Animals↗
PubMed2001

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.

Animals↗
PubMed1988

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase.

To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.

Aging↗
PubMed1985

Separation of mitochondrial creatine kinase (MiMi-CK) from cytosolic creatine kinase isoenzymes by Cibachrome-Blue affinity chromatography.

The mitochondrial isoenzyme of creatine kinase (MiMi-CK) was separated by affinity chromatography on Cibachrome-Blue-Sepharose (Sepharose-Blue, Pharmacia). While the soluble CK isoforms (BB-CK and MM-CK) were specifically eluted by raising the pH of the column buffer from pH 6.0 to pH 8.0, MiMi-CK remained bound under these conditions but was specifically eluted by subsequent addition of ADP to the pH 8.0 buffer. This one-step method allows a fast and efficient separation of MiMi-CK from MM-and BB-CK isoenzymes and at the same time an enrichment of MiMi-CK by about 50-fold. Since MiMi-CK can be assayed separately after isolation by affinity chromatography on Sepharose-Blue, this method may be of clinical importance.

Animals↗
PubMed1989

Immunocytochemical localization of creatine kinase M in canine myocardial cells: most creatine kinase M is distributed in the A-band.

The localization of creatine kinase (CK) M in canine myocardium was immunocytochemically studied by a direct immunoperoxidase method. Specific antiserum against CK-M was produced in rabbits immunized with canine CK-MM. An anti-CK-M Fab'-horseradish peroxidase conjugate was prepared by the maleimide method. Frozen sections prepared from fixed canine myocardium were stained with the conjugate and observed by light and electron microscopy. In light microscopy of longitudinal sections, CK-M showed a cross-striated pattern consisting of distinct broad and narrow brown bands. Immunoelectron microscopy revealed that the regions of the broad and narrow brown bands corresponded to the A-band and the Z-line, respectively. Most CK-M in the A-band was associated with the thick fibers, and a small amount of CK-M was found in the M-line. These findings suggest that ATP regeneration from the ADP produced by myosin ATPase is related to the participation of this CK associated with the thick fibers rather than that of the M-line-bound CK. Creatine kinase M was also found in the sarcolemmal membrane, the membranes of the sarcoplasmic reticulum, and the mitochondrial outer and inner membranes. This report provides new information for understanding the physiological role of the phosphorylcreatine shuttle in the myocardial energy transport system.

Animals↗
PubMed1986

High content of creatine kinase in chicken retina: compartmentalized localization of creatine kinase isoenzymes in photoreceptor cells.

Two isoforms of creatine kinase (CK; ATP:creatine N-phosphotransferase, E.C. 2.7.3.2), brain type (BB-CK) and mitochondrial type (MiMi-CK), but not the muscle types (MM- or hybrid MB-CK), were identified by cellulose polyacetate electrophoresis and immunoblots in retina from adult chickens. Indirect immunofluorescence labeling of cryosections of retinas revealed high concentrations of BB-CK in both rod and cone photoreceptor cells. Most of the fluorescence staining with anti-B-CK antibodies was found within the myoid and the ellipsoid portions of inner segments and the peripheral region of the outer segments. Significant staining with anti-B-CK antibodies was also found in horizontal cells and in the optical nerve fibers, with additional stratified staining in the inner plexiform layer. MiMi-CK was solely demonstrated in the ellipsoid portion of the photoreceptor cells. The presence of high concentrations of compartmentalized CK isoenzymes within photoreceptor cells (approximately equal to 30 enzyme units/mg) as well as the relatively high concentration of total creatine in these cells (approximately equal to 10-15 mM) indicates an important physiological function for CK and phosphocreatine in the energy transduction of vision.

Animals↗