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PubMed2000

Reactivation and refolding of reassociated dimers of rabbit muscle creatine kinase.

Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a good model for studying dissociation and reassociation during unfolding and refolding. This study compares self-reassociated CK dimers and CK dimers that contain hybrid dimers under proper conditions. Creatine kinase forms a monomer when denatured in 6 M urea for 1 h which will very quickly form a dimer when the denaturant is diluted under suitable conditions. After modification by DTNB, CK was denatured in 6 M urea to form a modified CK monomer. Dimerization of this modified subunit of CK occurred upon dilution into a suitable buffer containing DTT. Therefore, three different types of reassociated CK dimers including a hybrid dimer can be made from two different CK monomers in the proper conditions. The CK monomers are from a urea-denatured monomer of DTNB-modified CK and from an unmodified urea dissociated monomer. Equal enzyme concentration ratios of these two monomers were mixed in the presence of urea, then diluted into the proper buffer to form the three types of reassociated CK dimers including the hybrid dimer. Reassociated CK dimers including all three different types recover about 75% activity following a two-phase course (k1 = 4.88 x 10(-3) s(-1), k2 = 0.68 x 10(-3) s(-1)). Intrinsic fluorescence spectra of the three different CK monomers which were dissociated in 6 M urea, dissociated in 6 M urea after DTNB modification, and a mixture of the first two dissociated enzymes were studied in the presence of the denaturant urea. The three monomers had different fluorescence intensities and emission maxima. The intrinsic fluorescence maximum intensity changes of the reassociated CK dimers were also studied. The refolding processes also follow biphasic kinetics (k1 = 3.28 x 10(-)3 s(-1), k2 = 0.11 x 10(-3) S(-1)) after dilution in the proper solutions. Tsou's method [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381-436] was also used to measure the kinetic reactivation rate constants for the different three types of reassociated CK dimers, with different kinetic reactivation rate constants observed for each type. CK dissociation and reassociation schemes are suggested based on the results.

Animals↗
PubMed2002

Creatine kinase and creatine transporter in normal, wounded, and diseased skin.

Skin comprises many cell types that are characterized by high biosynthetic activity and increased energy turnover. The creatine kinase system, consisting of creatine kinase isoenzymes and creatine transporter, is known to be important to support the high energy demands in such cells. We analyzed the presence and the localization of these proteins in murine and human skin under healthy and pathologic conditions, using immunoblotting and confocal immunohistochemistry with our recently developed specific antibodies. In murine skin, we found high amounts of brain-type cytosolic creatine kinase coexpressed with lower amounts of ubiquitous mitochondrial creatine kinase, both mainly localized in suprabasal layers of the epidermis, different cell types of hair follicles, sebaceous glands, and the subcutaneous panniculus carnosus muscle. With exception of sebaceous glands, these cells were also expressing creatine transporter. Muscle-type cytosolic creatine kinase and sarcomeric mitochondrial creatine kinase were restricted to panniculus carnosus. Immediately after wounding of murine skin, brain-type cytosolic creatine kinase and a creatine transporter-subspecies were transiently upregulated about 3-fold as seen in immunoblots, whereas the amount of ubiquitous mitochondrial creatine kinase increased during days 10-15 after wounding. Healthy and psoriatic human skin showed a similar coexpression pattern of brain-type cytosolic creatine kinase, ubiquitous mitochondrial creatine kinase, and creatine transporter in this pilot study, with creatine transporter species being upregulated in psoriasis.

Animals↗
PubMed1982

Optimized bioluminescence assay of creatine kinase and creatine kinase B-subunit activity.

Spectrophotometry of CK-B subunit activity based on immunoinhibition is rapid and convenient. However, the low sensitivity limits clinical applications to serum samples with CK-B activities considerably above normal. A sensitive, but not optimized, bioluminescent assay of CK-B, also based on immunoinhibition, has been described. We improved this method by the use of an ADP concentration saturating the CK reaction and by the combined use of AMP and diadenosine pentaphosphate for inhibition of adenylate kinase. The optimized bioluminescent method is similar to the corresponding spectrophotometric method (reaction conditions, analytical procedure, numerical results, etc.) and results by the two methods correlate well. However, the measuring time is shorter and, more important, the sensitivity of the bioluminescent method allows accurate determinations of CK-B, even in serum from healthy individuals (CV approximately 3%). Kits and instrumentation (manual and automatic) for determination of CK and CK-B are now commercially available.

Adenylyl Cyclases↗
PubMed1994

Comparison of the value of novel rapid measurement of myoglobin, creatine kinase, and creatine kinase-MB with the electrocardiogram for the diagnosis of acute myocardial infarction.

OBJECTIVE: To determine whether serum myoglobin, creatine kinase, and creatine kinase-MB measured at admission by rapid, compact, and easy to use automated quantitative analysers (results within 10 min) helped the early identification of acute myocardial infarction. The results were compared with the data obtained from the electrocardiograms recorded at admission. DESIGN: A prospective study. SETTING: Coronary care unit. PATIENTS: 94 consecutive patients with suspected myocardial infarction. Myocardial infarction was subsequently confirmed in 44 patients and excluded in 50. METHODS: All admission serum myoglobin, creatine kinase, and creatine kinase-MB were measured by clinical staff using analysers in the coronary care unit. An admission electrocardiogram was obtained from all patients. RESULTS: The sensitivity, specificity, and predictive accuracy for diagnosing myocardial infarction were: electrocardiogram 68%, 100%, and 85%; myoglobin 57%, 100%, and 80%; creatine kinase (threshold of 190 U/l) 34%, 98%, and 68%; creatine kinase-MB (threshold of 25 U/l) 43%, 100%, and 73%. When the electrocardiographic and myoglobin data were combined the sensitivity improved to 91%, diagnostic accuracy to 96%, with specificity of 100%. The results for the electrocardiogram and creatine kinase-MB were 80%, 90%, 100% respectively and those for the electrocardiogram with creatine kinase were 80%, 89%, 98% respectively. CONCLUSIONS: Admission myoglobin, creatine kinase, and creatine kinase-MB measurements were not as useful as the electrocardiogram for the diagnosis of acute myocardial infarction. Combining the electrocardiogram and myoglobin data substantially improved the sensitivity and predictive accuracy for the diagnosis of acute myocardial infarction.

Blood Chemical Analysis↗
PubMed2006

Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase.

Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60 degrees C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A(lim)) is proportional to protein concentration at both temperatures. The first order rate constant (k(I)) does not depend on protein concentration in the range 0.05-0.2 mg/ml at temperature 50.6 degrees C, but at temperature 60 degrees C it increases with the growth of protein concentration in the range 0.1-0.4 mg/ml. Kinetic curves, shown in coordinates {A/A(lim); t}, in experiments at 50.6 degrees C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60 degrees C, half-transformation time t(1/2) = ln2/k(I) decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60 degrees C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6 degrees C aggregation rate is limited by the stage of protein molecule denaturation, but at 60 degrees C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation.

Animals↗
PubMed2001

The three-dimensional structure of cytosolic bovine retinal creatine kinase.

Creatine kinase (CK) catalyses the reversible transfer of the phosphate moiety from phosphocreatine (PCr) to ADP, generating creatine and ATP. The crystal structure of a cytosolic brain-type creatine kinase is reported at 2.3 A. The biological dimer sits on a crystallographic twofold axis. The N-terminal residues of both subunits come very close to the crystallographic twofold at the dimer interface. The electron density observed is consistent with two alternative conformations for the N-termini, as previously found for chicken brain-type creatine kinase.

Amino Acid Sequence↗
PubMed1976

An approach to the antigenic structure of arginine kinase and creatine kinase. Physical, chemical and immunological study of some modified derivatives.

The antigenic structure of arginine kinase and creatine kinase has been approached using chemical modifications and enzymatic cleavage. Mild performic oxidation that, with a restricted number of oxidized amino acid residues, results for both enzymes in a severe decrease of the helical structure and in a large increase of the protein-solvent interactions, affects differently their antigenic reactivity: when compared with antisera to the homologous native enzymes, arginine kinase and its oxidized derivative cross-react fully, while creatine kinase and its oxidized derivative cross-react only about 30%. The persistence of the antigenic reactivity of arginine kinase through drastic structural alterations is confirmed by the high inhibitory capacity (about 80%) of crude tryptic hydrolyzates towards the combination of argining kinase with its specific antibodies. Tryptic peptides of creatine kinase, obtained in the same conditions, inhibit weakly (about 12%) the homologous antigen-antibody interaction. The participation of the lysines in the antigenicity of arginine kinase and creatine kinase is suggested by the enhanced inhibitory capacity of the tryptic hydrolyzates when the cleavage is restricted to the arginyl peptide bounds, and was verified for arginine kinase through assays with lysine-modified derivatives.

Amino Acids↗
PubMed1982

Serum myoglobin, creatine kinase and creatine kinase-MB as mutually supportive indices of myocardial infarction and infarct size.

A comparison was made between the appearance of serum myoglobin and creatine kinase in 22 patients with acute myocardial infarction who were admitted to a coronary-care unit within four hours of onset of chest pain. The MB isoenzyme of creatine kinase was measured in 12 patients. The more rapid appearance and disappearance of myoglobin relative to creatine kinase and creatine kinase-MB was confirmed, as was the correspondence between their respective peak values. A significant correlation was also obtained between the area under the myoglobin time-course and the respective peak levels. Whereas creatine kinase activity declined exponentially from a single peak, myoglobin appeared in multiple episodes inadequately represented by a single peak value and having no clear clinical correlation. The role of myoglobin as a diagnostic aid in myocardial infarction is probably limited to its ability to support creatine kinase and creatine kinase-MB as indices of infarct size.

Creatine Kinase↗
PubMed1985

Isolation and characterization of acetylcholine receptor membrane-associated (nonreceptor v2-protein) and soluble electrocyte creatine kinases.

Creatine kinase has been identified as a most prominent component of Torpedo electric organ and a minority constituent of the acetylcholine receptor (AChR) membranes obtained therefrom. Purification by low temperature ethanol extraction, precipitation of the Mg2+-enzyme complex, and mercurial-agarose chromatography yield preparations of soluble kinase with specific activities greater than 550 units/mg protein. Retention times in ion-exchange high performance liquid chromatography, electrophoretic behavior, immunochemical properties, tryptic mapping, and amino acid composition enable the comparison of creatine kinase isoenzymes. The denatured subunits of the predominant species have pI values of 6.3-6.8 and Mr = 40,000-42,000 characteristic of the so-called v2 proteins and show cross-reactivity with antibodies against the BB ("brain" type) creatine kinase. The MM ("muscle" type) antigens could be detected in the total electrocyte, but not in the AChR membranes; they have a slightly lower molecular weight and higher pI. The in situ membrane association of the BB isoenzyme is confirmed by immunocytochemistry. The apparent Km values for the substrate creatine phosphate are 2.2 mM for the AChR membrane-associated enzyme and 2.5 mM for the muscle form. The apparent Km values for Mg2+-ADP are 0.54 and 0.22 mM, respectively. Thus, a 2-fold higher affinity in the binding of ADP to the binary enzyme-creatine-P complex results from membrane association.

Amino Acids↗
PubMed1990

Optimised function for determining time to peak creatine kinase and creatine kinase-MB as non-invasive reperfusion indicators after thrombolytic therapy in acute myocardial infarction.

STUDY OBJECTIVE: The aim of the study was to investigate the use of an optimised function to approximate and interpolate the time course of serum creatine kinase and creatine kinase-MB values after thrombolytic therapy in acute myocardial infarction. DESIGN: A three parameter interpolating function was developed which approximates the time course of serum enzyme levels. In the proposed function, time to peak creatine kinase and maximum of creatine kinase determined from raw data were used as starting parameters of the non-linear interpolation routine, thus providing ideal starting conditions for the iteration. The efficacy of the function was compared with that of three other functions cited in published reports (log-normal distribution function, modified gamma density function, three compartment function). SUBJECTS: Serum enzyme data from 20 patients with acute myocardial infarction were used in the comparisons. The patients have all been treated with anisoylated plasminogen streptokinase activator complex. RESULTS: In comparison with the other models, deviations of the experimental model function from the raw data were minimal. The fit remained stable for time intervals between blood samples of up to 6 h. CONCLUSIONS - Due to its numerical stability, the function outlined in this study is suitable for large clinical reperfusion trials. In the case of uncomplicated infarctions without thrombolytic therapy, the area under the creatine kinase activity curve could be directly calculated in terms of maximum activity and time to peak.

Anistreplase↗
PubMed1982

[Isoenzymatic forms and distribution of adenylate kinase and creatine kinase in the bovine adrenal medulla].

Adenylate kinase, creatine kinase as well as their substrate and product levels have been investigated in the adrenal medullary tissue. The concentration of adenine nucleotides and creatine + creatine phosphate are 12.6 +/- 0.4 and 6.9 +/- 0.4 mumol/g wet weight respectively. Adenylate kinase is mainly in the cytosol; only 4% was found in mitochondria. The cytosol enzyme presents a Km for AMP of 5 X 10(-4) M and a Ki for diadenosine pentaphosphate of 0.6 X 10(-6) M. In gel electrophoresis, only one band of adenylate kinase activity can be seen, and its mobility is different from that of the brain enzyme. Creatine kinase from adrenal medulla is mainly found in cytosol; only 3-4% was associated with mitochondria. The cytosolic enzyme is mainly the BB isozyme form.

Adenylate Kinase↗
PubMed2001

Changes in the mitochondrial proteome from mouse hearts deficient in creatine kinase.

Creatine kinase (CK) is an abundant enzyme, important for maintenance of high-energy phosphate homeostasis in many tissues including heart. Double-knockout CK (DbKO-CK) mice missing both the muscle (MM) and sarcomeric mitochondrial (ScMit) isoforms of CK have recently been studied. Despite a large change in skeletal muscle function in DbKO-CK mice, there is little functional change in the heart. To investigate whether there are specific changes in cardiac mitochondrial proteins associated with the loss of MM- and ScMit-CK isoforms, we have used difference gel electrophoresis (DIGE) to compare mitochondrial proteins from wild-type and DbKO-CK mice. Mass spectrometry fingerprinting was used to identify 40 spots as known mitochondrial proteins. We have discovered that the loss of MM- and ScMit-CK isoforms did not cause large scale changes in heart mitochondrial proteins. The loss of ScMit-CK was readily detected in the DbKO-CK samples. We have also detected a large decrease in the precursor form of aconitase. Furthermore, two mitochondrial protein differences have been found in the parent mouse strains of the DbKO-CK mice.

Aconitate Hydratase↗
PubMed1999

Crystallization and preliminary X-ray analysis of human muscle creatine kinase.

Creatine kinase is a key enzyme in the energy homeostasis of cells and tissues with high and fluctuating energy demands. Human muscle MM creatine kinase is a dimeric protein with a molecular weight of approximately 43 kDa for each subunit. It has been crystallized by the hanging-drop vapor-diffusion method using 2-methyl-2, 4-pentanediol as precipitant. The crystals belong to the enantiomorphous space group P6222 or P6422 with cell parameters of a = b = 89.11 and c = 403.97 A. The asymmetric unit of the crystal contains two subunits. A data set at 3.3 A resolution has been collected using synchrotron radiation.

Creatine Kinase↗
PubMed1998

Purification, crystallization and preliminary crystallographic analysis of bovine cytosolic brain-type creatine kinase.

Creatine kinase (E.C. 2.3.7.2) is an important enzyme in energy metabolism which catalyzes the reversible transfer of a phosphoryl group between phosphocreatine and ADP to give ATP. Large quantities of a brain-type creatine kinase have been isolated from bovine photoreceptor cells and crystals suitable for X-ray diffraction analysis have been obtained by hanging-drop vapor diffusion. Crystals grow as tetragonal bipyramids in space group P43212 with cell dimensions a = b = 96.49, c = 108.42 A and diffract to at least 2.7 A resolution.

Animals↗