Isoenzymes of creatine kinase

In resting muscle the high concentration of ADP does not decrease the proton gradient effectively and the high membrane potential slows electron transport. ADP, formed when ATP is hydrolyzed by myosin ATPase during contraction, may stimulate electron transport. However, the concentration of ATP (largely as its Mg salt) is buffered by its readily reversible formation from creatine phosphate catalyzed in the intermembrane space, and in other cell compartments, by the various isoenzymes of creatine kinase (reviewed by Walliman et al., 1992). 

Both aspartate aminotransferase and alanine aminotransferase are released into the blood after damage to tissues or after cell death. Consequently, they are used as diagnostic tools when heart or liver damage has occurred, such as after a heart attack or in hepatitis, respectively. Other enzymes are also released into the blood at such times. For example, damage to heart muscle is further characterized by the presence of isoenzymes of creatine kinase or lactate dehydrogenase in the plasma. 

Creatine kinase (CK) occurs in high concentrations in the brain, cardiac and skeletal muscle and is elevated in the blood with muscle damage. A rise in CK is seen in acute myocardial infarction but also in other conditions. A more specific marker is creatine kinase MB (CK-MB), which is an isoenzyme of creatine kinase that is more specific for cardiac muscle damage. CK or CK-MB will rise approximately 4 hours after an acute cardiac event and will reach a peak after approximately 24 hours and will remain raised for 3-4 days. 

Salbutamol causes an increase in the activity of the MB isoenzyme of creatine kinase, which has been interpreted as meaning that it might be cardiotoxic (3). 

E620 Hadberg, A., Hassager, C., Hildebrandt, P. and Christiansen, C. (1990). Comparison of two immunoinhibitory methods with agarose gel-electrophoresis for measuring the MB isoenzyme of creatine kinase in serum from cases of suspected myocardial infarction. J. Clin. Chem. Clin. Biochem. 28, 453-458. 

ST77 Bermes, E.W., Kahn, S.E., Jandresici, M.A., Augustine, G. and Khan, I. (1990). Analytical and clinical comparison of four methods for the determination of the MB isoenzyme of creatine kinase. Clin. Chem. 36, 1130, Abstr. 831. 

Contact sites were first described by Hacken-BROCK (1968) in thin sections of liver mitochondria as places where the inner and outer mitochondrial membranes were in very close apposition. Van Ve-NETiE and Verkleij (1982) and Knoll and Brdiczka (1983) characterized them in freeze-fractured mitochondria. Knoll and Brdiczka (1983) and Brdiczka et al. (1986) postulated that contact sites play an important role in the regulation of the mirochondrial metabolism. Under nor-moxic conditions, the ATP formed in the mitochondria is converted into creatine phosphate by the activity of the translocase, and the mitochondrial isoenzyme of creatine kinase (Wallimann et al. 1992). So, if the cardiac metabolism is stimulated the mitochondrial ATP formation increases, as does the mitochondrial creatine kinase. Since mitochondrial creatine kinase is active in mitochondrial contact sites (Biermans et al. 1990, Nicolay et al. 1990, Jacob et al. 1992), and can even induce contact site formation (Rojo et al. 1991), the surface density of mitochondrial contact sites in this situation will be high. Mitochondria lose the ability to form contact sites after more than 15 min of ischaemia and this might be a first indication of irreversible injury (Barker et al. 1995). 

Jacobs, H., Heldt, H. W., and Klingenberg, M., 1964, High activity of creatine kinase in mitochondria from muscle and brain and evidence for a separate mitochondrial isoenzyme of creatine kinase, Biochem. Biophys. Res. Commun. 16 516. 

Wallimann, T., Wyss. M., Brdicza, D., Nicolay, K.. Eppenberger, H.M. (1992). Intracellular compartmentation. structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands The phosphocreatine circuit for cellular energy homeostasis. Biochem. J. 281,21-40. 

Jockers, W. Wretouy, E. Pfleiderer, 6. Quantitation of creatine kinase isoenzymes in human tissues and sera by an immunological method. Clin. Chim. Acta. (1975),... 

New manual and automated method for determining activity of creatine kinase isoenzyme MB by use of dithiothreitol Clinical applications. Clin. Chem. (1975), 1612-1618. 

Figure 6, High pressure liquid chromatogram of creatine kinase isoenzymes. First peak, MM second peak, BB. Conditions 50 cm X 4.8 mm (i.d.) column with yydac porous layer bead anion exchange mobile phase, step gradient Solvent A, 10 mmol/liter Tris buffer, pH 8.3 solvent B, 10 mmol/liter Tris buffer, pH 7.0,0.5 mol KCl flow rate, 2 ml/min detection, collected fractions assayed (45).

In the course of studying the mechanism of action of creatine kinase from rabbit skeletal muscle (M.M isoenzyme), Kenyon and coworkers (4,90) have been involved in the design of specific irreversible inhibitors that are active-site-directed (affinity labels). Creatine kinase catalyzes the reversible transfer of a phosphoryl group ( the elements of "POi") from ATP to creatine, as shown in the following reaction ... 

Subunit structure and electrophoretic mobility and enzyme activity of creatine kinase isoenzymes. 

The patient, a 63-year-old Caucasian female, was hospitalized on 4 April 2002 though 10 April 2002 for a non-ST segment elevation myocardial infarction (non-Q-wave MI per chart documentation). She had a negative adenosine stress test after the initial event. Her serum cardiac-specific troponin I (cTnl) concentration 24 hours after her onset of chest pain was 1.4 pg/L (upper limit of normal is 0.3 ng/mL), and her creatine kinase (CK) MB level was 12.5 pg/L (upper limit of normal 6.0 ng/mL). Three days post-event her cTnl level was 0.5 pg/L and her CK-MB level was 4.5 pg/L (Fig. 5-1). MB refers to one of the isoenzyme forms of CK found in serum. The form of the enzyme that occurs in brain (BB) does not usually get past the blood-brain barrier and therefore is not normally present in the serum. The MM and MB forms account for almost all of the CK in serum. Skeletal muscle contains mainly MM, with less than 2% of its CK in the MB form. MM is also the predominant myocardial creatine kinase and MB accounts for 10%-20% of creatine kinase in heart muscle. 

Although it is true that abnormal proteins increase with age, most of them are a result of posttranslational changes. An example is the various isoforms of creatine kinase (CK). Here, the major isoenzyme, CK-MM (isoform CK-33), is normally synthesized in the heart and skeletal muscle. However, after its release into the circulation, carboxypeptidase hydrolyzes the terminal lysine from one of the M-peptides to form CK-32. Subsequent hydrolysis of the terminal lysine from the second M-peptide produces the third isoform, CK-3i (W8). Numerous similar posttranslational proteins are produced. Hence, the presence of abnormal proteins per se does not support this aging theory. 

Sket, D., Brzin, M., Vreca, I. (1989). Effect of HI-6 and diazepam on the increase of creatine kinase isoenzymes activity in plasma of atropinized, soman-poisoned rats. Acta Pharm, Jugoslav. 39 151-9. 

VoL LVn [6]. Determination of Creatine Kinase Isoenzymes in Human Serum by an Immunological Method Using Purified Firefly Luciferase. A. Lundin. 

A 47-year-old man presented with severe myalgia and a raised creatine kinase MM isoenzyme (from skeletal muscle) while taking salbutamol from a multidose pressurized inhaler (SEDA-21, 182). The MM creatine kinase activity returned to normal when he stopped salbutamol and rested. When salbutamol was taken by inhaler or orally, the enzyme activity rose. When salbutamol was combined with exercise, even higher concentrations of creatine kinase resulted. A muscle biopsy specimen was consistent with a myopathy. 

ST79 Chapelle, J.-P. and El Allaf, M. (1990). Automated quantification of creatine kinase MB isoenzyme in serum by radial partition immunoassay, with use of the Stratus analyzer. Clin. Chem. 36, 99-101. 

ST92 Lianidou, E.S., Christopoulos, T.K. and Diamandis, E.P. (1990). Assay of creatine kinase isoenzyme MB in serum with time-resolved immunofluorom-etry. Clin. Chem. 36, 1679-1683. 

Methods for the Separation and Quantification of Creatine Kinase Isoenzymes... 

Panteghini M. Serum isoforms of creatine kinase isoenzymes. Clin Biochem 1988 21 211-8. 

Three cytosolic isoenzymes (CK-3, CK-2, CK-1) and one mitochondrial isoenzyme (CK-Mt) of creatine kinase (CK) (MW 80,000 Da for all 4 isoenzymes) have been identified and are easily separated on agarose and cellulose acetate by electrophoresis (see Chapter 21). Three different genes have been identified that encode for and are specific for CK-M, CK-B, and mitochondrial CK subunits.Although CK-3 (CK-MM) is predominant in both heart and skeletal muscle, CK-2 (CK-MB) has been shown to be more specific for the myocardium, which contains 10% to 20% of its total CK activity as CK-MB, compared with amounts varying from 2% to 5% in skeletal muscle. 

Abendschein DR, Fontanet HL, Nohara R. Optimized preservation of isoforms of creatine kinase MM isoenzyme in plasma specimens and their rapid quantification by semi-automated chromatofocusing. Clin Chem 1990 36 723-7. 

The isoenzyme pattern of creatine kinase in normal and diseased muscle has been investigated by several workers. Two subunits are recognized, usually denoted M and B the enzyme is a dimer, hence three forms are possible MM, BB, and the hybrid MB. Most muscles contain largely MM with sometimes a proportion of BB (the latter is characteristic of brain). Normal fetal muscle, however, contains a preponderance of BB (Gil, S3). After denervation, there is a relative decrease in MM (S3). In muscle from patients with fascioscapulohumeral dystrophy and polymyositis there was an increase in MB relative to MM, but a normal ratio was found in Duchenne and limb-girdle dystrophies and in most cases of neurogenic atrophy BB occurred in some cases of myotonic dystrophy (Gll). 

D7. Denton, M. S., Bostick, W. D., Dinsmore, S. R., and Mrochek, J. E., Continuously referenced, on-line monitoring of creatine kinase and lactate dehydrogenase isoenzymes for use in clinical diagnostics. In "Biological/Biomedical Applications of Liquid Chromatography H (G. L. Hawk, ed.), pp. 165-191. Dekker, New York, 1979. 

K18. Kudirka, P. J., Busby, M. G., Carey, R. N., and Toren, E. C., Jr., Separation of creatine kinase isoenzymes by high-pressure liquid chromatography. Clin. Chem. 21, 450-452 (1975). 

U. Wuerzburg, Measurement of Creatine Kinase Isoenzyme Activity by Immunological Methods, in Creatine Kinase Isoenzymes Pathophysiology and Clinical Application (ed. H. Lang), Springer-Verlag, New York, 1981, p. 49. 

Immobilized enzyme microreactor for the 561 continuously-referenced monitoring of isoenzyme activities of creatine kinase after liquid-chromatographic separation Evaluation of the analytical performance of 562 the enzyme-containing coils, adapted for use with the Technicon SMAC analyser in the determination of D-glucose... 

Wallimann T, Wyss M, Brdiczka D, Nicollay K, Eppenberger HM. Significance of intracellular compartmentation, structure and function of creatine kinase isoenzymes for cellular energy homeostasis The Phospho-Creatine Circuit . Biochem J 1992 281 21-24... 

Askenasy, N. and A.P. Koretsky, Differential effects of creatine kinase isoenzymes and substrates on regeneration in livers of transgenic mice. Am J Physiol, 273 (Pt 1) ... 

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