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PubMed1986

The isoelectric focusing of creatine kinase variants: I. The heterogeneity of creatine kinase in human heart cytosol and mitochondria.

Creatine kinase isoenzymes in cytosolic and mitochondrial fractions from human cardiac tissues were studied by analytical and preparative isoelectric focusing (IEF), electrophoresis and immunoinhibition. Analytical IEF on agarose gels revealed many creatine kinase variants in human cardiac cytosol prepared by extraction with a hypotonic medium. The bands located at approximately pH 5.5 were shown to contain creatine kinase-MB and minute creatine kinase-BB bands by electrophoresis. Two bands which focused closely together in IEF (pH 6.85-7.0) showed an electrophoretic migration pattern similar to creatine kinase-MM. One of them (IP 6.85) showed a complete inhibition by anti-creatine kinase-M antibodies, whereas the other showed only 50% inhibition. Increasing the salt concentration of tris-HCl (0.1 mol/l) in the extraction medium resulted in additional creatine kinase variants. They were characterized by high alkaline isoelectric points and were not inhibited by anti-creatine kinase-M antibodies. These variants corresponded to two cathodic bands in electrophoresis. The treatment of washed mitochondria with phosphate buffer resulted in a release of mitochondrial variants with different isoelectric points, as shown by analytical IEF in agarose gels. The same pattern was obtained by using preparative IEF. Variants with high alkaline isoelectric points gave rise to two cathodic bands upon electrophoresis. These two bands resembled those present in cytosol after extraction with high salt concentration. No complete inhibition with anti-creatine kinase-M was observed in any of the eluates. The mitochondrial variants exhibited different affinities towards creatine phosphate and ADP. Variants with higher alkaline isoelectric points showed lower Km-values for these substrates than those with less alkaline isoelectric points.

Creatine Kinase↗
PubMed1988

Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships.

The mitochondrial isoform of creatine kinase (Mi-CK, EC 2.7.3.2) purified to homogeneity from chicken cardiac muscle by the mild and efficient technique described in this article was greater than or equal to 99.5% pure and consisted of greater than or equal to 95% of a distinct, octameric Mi-CK protein species, with a Mr of 364,000 +/- 30,000 and an apparent subunit Mr of 42,000. The remaining 5% were dimeric Mi-CK with an apparent Mr of 86,000 +/- 8,000. Octamerization was not due to covalent linkages or intermolecular disulfide bonding. Upon dilution into buffers of low ionic strength and alkaline pH, octameric Mi-CK slowly dissociated in a time-dependent manner (weeks-months) into dimeric Mi-CK. However, the time scale of dimerization was reduced to minutes by the addition to diluted Mi-CK octamers of a mixture of Mg2+, ADP, creatine and nitrate known to induce a transition-state analogue complex (Milner-White, E.J., and Watts, D. C. (1971) Biochem. J. 122, 727-740). The conversion was fully reversible, and octamers were reformed by simple concentrations of Mi-CK dimer solutions to greater than or equal to 1 mg/ml at near neutral pH and physiological salt concentrations in the absence of adenine nucleotide. After separation of the two Mi-CK species by gel filtration, electron microscopic analysis revealed uniform square-shaped particles with a central negative-stain-filled cavity in the octamer fractions and "banana-shaped" structures in the dimer fractions. Mi-CK was localized inside the mitochondria by immunogold labeling with polyclonal antibodies. A dynamic model of the octamer-dimer equilibrium of Mi-CK and the preferential association of the octameric Mi-CK form with the inner mitochondrial membrane is discussed in the context of regulation of Mi-CK activity, mitochondrial respiration, and the CP shuttle.

Animals↗
PubMed2000

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.

Animals↗
PubMed1986

The isoelectric focusing of creatine kinase variants: II. The heterogeneity of creatine kinase in human serum with normal and elevated catalytic concentrations.

An effective and reliable method for the quantitative estimation of creatine kinase-MB, creatine kinase-MM variants and mitochondrial forms of creatine kinase in serum is presented. The high resolving power of isoelectric focusing allows the use of tetrazolium salts and meldola blue for the quantitative measurement without interfering non-specific reduction. The addition of thiol compounds to the agarose medium increases the sensitivity of the method, due to the inhibition of sulfhydryl group oxidation, and prevents enzyme degradation, which is a possible cause of an artificial heterogeneity. Depending upon the type of muscle and the degree of cell damage, we found 3-4 creatine kinase-MM sub-bands in sera with activities below 80 U/l. At elevated creatine kinase activities 3-11 creatine kinase-MM sub-bands were found. The appearance of creatine kinase-MB in serum indicates that damage has occurred to certain organs, especially the cardiac muscle. An organ with moderate or massive cell damage could release, in addition to the sarcoplasmatic creatine kinase variants, other forms with more alkaline isoelectric points (mitochondrial creatine kinase). The presence of such bands in serum of patients correlates with poor prognosis. Besides the separation of creatine kinase-MM sub-bands, creatine kinase-MB, creatine kinase-BB and of macroforms 1 and 2, the advantage of this method is the detection of mitochondrial creatine kinase forms, which in cellulose acetate electrophoresis migrate with creatine kinase-MM.

Catalysis↗
PubMed1995

Measurement of serum troponin T, creatine kinase MB isoenzyme, and total creatine kinase following arduous physical training.

We have compared measurement of cardiac troponin T by enzyme linked immunosorbent assay with creatine kinase MB isoenzyme (CK-MB) concentration measurement in 219 Royal Marine commandos with no evidence of cardiovascular disease who have elevated creatine kinase (CK) produced by arduous physical training. CK was elevated up to 22.6 times and CK-MB mass up to 6.6 times the upper reference limit. Only two commandos had detectable cardiac troponin T, with neither exceeding the upper reference limit of 0.2 micrograms/L. At decision thresholds optimized for diagnosis of acute myocardial infarction in previous published work, 58.3% of the total CK activity, 13.8% of the CK-MB concentration/CK activity ratio and 1.6% of CK-MB concentration measurements showed elevated values but no elevations in cardiac troponin T occurred. Cardiac troponin T is currently the investigation of choice for the differential diagnosis of patients with an elevated CK due to skeletal muscle trauma to exclude myocardial damage.

Aspartate Aminotransferases↗
PubMed1986

A new approach for the enzymatic estimation of infarct size: serum peak creatine kinase and time to peak creatine kinase activity.

The relations of several creatine kinase (CK) variables to angiographic left ventricular ejection fraction and abnormally contracting segments in the chronic phase were examined in 2 groups of patients with a first anterior acute myocardial infarction. In group A (n = 22), emergency coronary angiography was performed and nonsurgical early reperfusion was attempted. Such an early revascularization, which was considered partially present in group B (n = 16), which received conventional therapy, shifted the CK time-activity curve to the left and altered its relation to angiographic cardiac function. At similar levels of peak CK, myocardial damage was significantly smaller in patients with successful thrombolysis than in those with unsuccessful reperfusion and conventional therapy (p less than 0.01). In patients whose infarct was considered to be moderate according to peak CK (1,000 to 3,000 U/liter), there was significant correlation between time to peak CK and left ventricular ejection fraction or percent abnormally contracting segments irrespective of their group. The results suggest that one should take into account rapid washout and shorter time to peak CK when estimating enzymatic infarct size in humans. The multivariate analysis of cardiac function with peak CK and time to peak CK resulted in a closer correlation in all patients. Such a correction in the time to peak CK may be a clinically useful approach for better interpretation of infarct size.

Aged↗
PubMed1980

Cryoenzymic studies on the transition-state analog complex creatine kinase . ADPMg . nitrate . creatine.

Concomitant with the formation of the transition state analog complex creatine kinase . ADPMg . nitrate . creatine [Milner-White, E. J. and Watts, D. C. (1971) Biochem. J. 122, 727--740] there results a large difference spectrum. The shape of the spectrum was characteristic of tryptophan exposure. The kinetics of formation and final amplitude of the spectrum were studied at -15 degrees C using a stopped-flow apparatus. The results obtained allowed for a plausible reaction pathway for nitrate fixation: the ordered addition of nitrate and creatine to the binary enzyme-ADPMg complex, a slow protein isomerization and the final addition of a molecule each of nitrate and creatine. The kinetic parameters for the formation of the transition state analog complex were compared with those already known for the overall reaction obtained under the same conditions [Barman, T. E., Brun, A. and Travers, F. (1980) Dur. J. Biochem. 110, 397--403]. This comparison revealed a striking analogy between the two processes. This suggests that the conformation of creatine kinase in the analog complex is very similar to that in the transition state complex on the catalytic pathway.

Adenosine Diphosphate↗
PubMed1981

The creatine kinase curve area and peak creatine kinase after acute myocardial infarction: usefulness and limitations.

We determined creatine kinase (CK) curve areas in 112 patients with acute myocardial infarction. Two-hour sampling was performed for the first 24 hours or until peak CK was reached, and a gamma density function was used to calculate curve areas from all available samples. Attempts to predict CK curve area by means of the portion of the curve prior to peak CK proved to be inaccurate; not until values 2 hours or more beyond peak CK were utilized did predicted and actual CK areas agree well. A good correlation (r = 0.93) was found between CK area and peak CK. To establish an approach for detecting peak CK in the clinical setting, a range of sampling intervals (4 to 24 hours) was assessed; 4- and 6-hour sampling intervals for 48 hours produced maximum CK values at or above 85% of true peak CK in 90% and 89% of patients, respectively, and average maximum CK at both sampling intervals exceeded 94% of that obtained with 2-hour samplings. We conclude that this simplified approach can provide a basis for estimating infarct severity in the individual patient.

Creatine Kinase↗
PubMed1981

Macro creatine kinase BB: evidence for specific binding between creatine kinase BB and immunoglobulin G.

In the sera of four female patients with proven IgG-linked creatine kinase BB (macro creatine kinase BB) we studied the nature of the binding between creatine kinase isoenzymes and immunoglobulin G. The specificity towards the isoenzyme bound, the recombination of the complex after treatment with acid buffer, the site of the binding between both partners and an apparently monoclonal nature of the immunoglobulin G involved in the complex indicate that a specific antigen-antibody reaction is responsible for the existence of macro creatine kinase BB in the sera investigated.

Antigen-Antibody Complex↗
PubMed1987

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.

Animals↗
PubMed1988

Use of creatine kinase MB isoenzyme for diagnosing myocardial infarction when total creatine kinase activity is high.

The usefulness of measuring creatine kinase MB isoenzyme for diagnosing myocardial infarction when activities of total creatine kinase are very high is unclear. We conducted a retrospective study in an urban hospital that serves a largely indigent population. We concentrated on 146 patients whose creatine kinase activity was greater than 1000 U/L (upper limit of normal: 165 U/L for women and 225 U/L for men), with MB isoenzyme greater than 10 U/L and less than 5% of total creatine kinase. The positive predictive value of MB isoenzyme (isoimmune method) values greater than 10 U/L was between 11.6% and 56.8% when the value for total creatine kinase exceeded 1000 U/L. Using different values (MB greater than 4% of total creatine kinase) as positive for myocardial infarction would have resulted in far fewer false-positives, but 10 cases of myocardial infarction would have been missed. The most appropriate cutoff value for MB isoenzyme in this population (total creatine kinase greater than 1000 U/L) was found to be greater than 2% of total creatine kinase.

Adolescent↗
PubMed1976

Radioimmunoassay for creatine kinase isoenzymes.

Creatine kinase has three isoenzymes designated MM, MB, and BB, with BB being the grain form and MM the muscle form. Antibodies to BB creatine kinase were obtained by immunization of rabbits with human BB creatine kinase. The antibodies demonstrated specificity for BB and MB creatine kinase (myocardial isoenzyme) but no cross-reactivity with MM creatine kinase. With the use of this antibody, a highly sensitive radioimmunoassay capable of measuring picomolar amounts of MB creatine kinase has been developed. Clinical application of this method should provide a sensitive and specific test for the diagnosis of myocardial infarction.

Brain↗
PubMed1984

Purification and characterization of naturally occurring and in vitro induced multiple forms of MM creatine kinase.

MM creatine kinase purified from tissue exists as a single form but upon release into the plasma exhibits three forms which, based on increasing anodal migration on polyacrylamide gel electrophoresis, are referred to as MM1, MM2, and MM3. The three forms were isolated in pure form from plasma in sufficient quantities for biochemical and physiological studies. Chromatofocusing results show that MM1, MM2, and MM3 have distinct isoelectric points of 7.58, 7.43, and 7.30, respectively. Analysis on high pressure liquid chromatography showed MM1 and tissue creatine kinase (MMt) to have identical peptide maps, but MM2 and MM3 were different in at least one amino acid. Tissue MM creatine kinase was converted in vitro to MM2 and MM3 after incubation in plasma. Conversion was unaffected by nonspecific proteases but inhibited by benzoylarginine, epsilon-amino caproic acid, and guanidinoethylmercaptosuccinic acid, specific inhibitors of carboxypeptidase N. Digestion of purified tissue MM creatine kinase with carboxypeptidases N and B produced MM2 and MM3 which were identical with the forms produced in vivo. Hybridization experiments with MM1 plus MM3 produced all three forms, as did tissue MM creatine kinase plus MM2. MM2 was also able to produce all three forms in the experiments. The plasma half-life of tissue MM creatine kinase or MM1 was shown to be about one-half that of MM3. The results suggest that hydrolytic cleavage of a basic amino acid, presumably by carboxypeptidase N, is responsible for conversion of MMt to MM2 and MM3. COOH-terminal amino acid analysis demonstrated the COOH-terminal amino acid of canine and rabbit MM1 to be lysine. Thus, we conclude that cleavage of the COOH-terminal lysine from MMt by serum carboxypeptidase N generates two additional MM creatine kinase isoforms with prolonged plasma clearance rates.

Animals↗