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ABSTRACT: Creatine kinase (CK) isoenzymes MM, MB, and BB are located primarily in the cell cytosol, and increased CKMB in plasma is the hallmark of myocardial infarction. However, whether CK is released with reversible ischemic injury remains controversial. Here, we assessed plasma CK activity--cytosolic and mitochondrial CK--in serial samples (every 10 min for 60 min, then hourly or every 4 h for 48 h) from 46 conscious dogs after transient or sustained coronary occlusion. Four dogs were sham-operated (controls); four underwent sustained coronary occlusion (96 h); and 38 underwent transient coronary occlusion (10-40 min) followed by 48 h of reperfusion. In postmortem histological examination of the dogs' hearts by light and electron microscopy, we looked for ischemia or necrosis. The presence of cell swelling and glycogen depletion was indicative of ischemia, whereas the added presence of cell disruption indicated necrosis. Coronary occlusion for > or = 20 min consistently increased plasma mitochondrial and total CK activity and produced histologically evident myocardial necrosis. In contrast, after 10 to 15 min of coronary occlusion, 12 of 14 animals, despite extensive severe reversible ischemia, showed no increase in plasma CK; the remaining 2, which had increased plasma CK, had subendocardial necrosis. Thus, cytosolic or mitochondrial CK is released from the heart only when there has been irreversible myocardial injury-a finding with significant diagnostic and therapeutic implications.
Clinical Chemistry 04/1997; 43(3):467-75. · 7.91 Impact Factor
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ABSTRACT: Isoforms (derived from the same isoenzyme but distinguished by differences in isoelectric point) of MM creatine kinase appear in plasma after myocardial infarction. They are formed by conversion of the tissue form of creatine kinase (MM-A, pI 7.80) to progressively more acidic species (MM-B, pI 7.50) and MM-C (pI 7.20) after release into the circulation. To define the changes responsible for myocardial MM creatine kinase isoform formation in humans and dogs, purified isoforms were treated with trypsin or cyanogen bromide. The digests were fractionated by reverse-phase high pressure liquid chromatography. Comparison of proteolytic maps showed that MM-A and MM-C were each characterized by a single, unique peptide peak. Maps of MM-B creatine kinase contained both of these peaks. Sequence analysis and comparison with the complete amino acid sequence of MM creatine kinase showed that the peptide unique to MM-A corresponded to the COOH-terminal tryptic or CNBr peptide. The peptide unique to MM-C was shown to have the same amino acid composition except for lysine (the COOH-terminal amino acid). Thus, isoform formation is characterized by the successive removal of the COOH-terminal lysine residue from one M subunit at a time resulting in the conversion of MM-A to isoforms MM-B and MM-C.
Journal of Biological Chemistry 01/1986; 260(28):14988-92. · 4.77 Impact Factor
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ABSTRACT: To quantify individual isoforms of canine myocardial MM creatine kinase (CK) we developed an immunoblot procedure suitable for analysis of large numbers of samples. Isoforms in plasma were separated by agarose electrophoresis, immobilized on nitrocellulose, detected with anti-MM followed by a labeled second antibody, and quantified by well counting. The amount of non-CK protein was reduced with sequential ethanol precipitation. Reproducibility of the assay developed was high with a standard deviation of 5.9% of mean values for each isoform. Variation of results after serial dilutions of samples constituted with 33.3% of each of the three isoforms was modest with a standard deviation of 3.5%. The method developed provided sensitive, discrete resolution of individual MM CK isoforms under nondenaturing conditions and should prove useful for improved, objective definition of the time of onset of irreversible injury to myocardial tissue rendered ischemic and hence improved stratification of patients studied in clinical trials concerned with protection and salvage of myocardium.
Analytical Biochemistry 09/1985; 149(1):209-17. · 3.00 Impact Factor
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ABSTRACT: Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at Vmax. The values for Km (mM/L) derived from Lineweaver-Burke plots are shown: (Table: see text). The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.
Molecular and Cellular Biochemistry 08/1985; 67(2):151-9. · 2.06 Impact Factor
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ABSTRACT: Subforms of creatine kinase, moieties derived from the same isoenzyme but exhibiting slightly different isoelectric points (isoforms), appear in plasma after release of CK isoenzymes from myocardium undergoing infarction. To determine whether isoform patterns in plasma permit precise dating of the onset of initial and recurrent infarction, it is first necessary to characterize the kinetics of isoform interconversion and to ascertain whether one dominant form is present in myocardium prior to release of each tissue isoenzyme. Accordingly, we developed a nondenaturing procedure for quantification of MM CK isoforms and analyzed tissue isoform content and kinetics of isoform interconversion in plasma in vivo and in vitro. MM CK in canine myocardium was found to consist of predominantly one isoform (95%), MMA (pI = 7.91). When purified MMA (420 IU/kg) was injected intravenously in conscious dogs, two isoforms, MMB (pI = 7.74) and MMC (pI = 7.51), appeared with a consistent temporal pattern, and MMA disappeared from plasma within 8 hours, with a disappearance rate three times greater than that of total MM CK activity. Incubation of MMA in vitro at 37 degrees C with canine plasma in concentrations comparable to those after intravenous administration in vivo resulted in a similar temporal pattern of appearance of MMB and MMC and disappearance of MMA with kinetics correlating closely with those in vitro (for MMA disappearance r = 0.985, and for MMC appearance r = 0.986). Incubation of purified MMB and MMC with plasma demonstrated that the conversion of MMA to MMB and to MMC was sequential and unidirectional. Specific activity (international units per milligram immunoassayable protein) was the same for all three isoforms. These results indicate that several conditions necessary for delineation of the chronology of infarction by isoform analysis are fulfilled and that kinetics of interconversion of isoforms in vivo are paralleled in vitro.
Journal of Laboratory and Clinical Medicine 04/1984; 103(3):470-84. · 2.62 Impact Factor
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ABSTRACT: Purification of human mitochondrial creatine kinase has been difficult and procedures that were highly successful in purifying canine enzyme failed for human mitochondrial creatine kinase. In the present study, we employed ultracentrifugation to remove the lipid, urea to prevent aggregation, followed by a final step of chromatofocusing which yielded a preparation of human mitochondrial creatine kinase with a specific enzyme activity of greater than 400 IU/mg. Biochemical and immunological characterization showed the preparation to be highly pure and free of even trace amounts of other creatine kinase isoenzymes. Antiserum specific for mitochondrial creatine kinase was developed which exhibited no cross-reactivity to cytosolic creatine kinase and mitochondrial creatine kinase did not cross-react with antiserum to the cytosolic forms. Marked differences were noted, both biochemically and immunologically, between mitochondrial creatine kinase and the cytosolic forms. Human mitochondrial creatine kinase was shown to have a molecular weight of around 82,000 and to be composed of two subunits of equal molecular weights around 41,000. Aggregates of mitochondrial creatine kinase were observed with molecular weights of around 200,000 in the absence of urea or if isolated from material after having undergone proteolysis. Isolation from fresh material or in the presence of urea inhibited aggregate formation for both canine and human mitochondrial creatine kinase. Despite claims of several investigators that mitochondrial creatine kinase exhibits two to three forms with varying molecular weights, our data indicate a single enzyme form made up of a subunit with a molecular weight of 41,000 and the high molecular weight aggregates appear to be induced artifacts. A radioimmunoassay was developed for human mitochondrial creatine kinase which, with appropriate modifications, should detect mitochondrial creatine kinase in human plasma.
Journal of Biological Chemistry 01/1984; 258(24):15346-54. · 4.77 Impact Factor
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ABSTRACT: Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.
Journal of Biological Chemistry 05/1980; 255(7):2870-7. · 4.77 Impact Factor
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ABSTRACT: The MM isoenzyme of creatine kinase, a dimer composed of two M ("muscle type") subunits, is found in myocardium, where it constitutes 85% of tissue CK, and in skeletal muscle, where it constitutes virtually 100%, as well as in other tissues. The tissue form is designated MMA. When MMA circulates in plasma, it undergoes stepwise, post-translational modification, mediated by proteolytic enzymes in plasma and giving rise to isoforms called MMB and MMC, which lack carboxy terminal lysine on one or two subunits, respectively. We have shown previously that changes with time in plasma profiles of MM creatine kinase (CK) isoforms in dogs reflect myocardial infarction within 1 hour after the onset of coronary occlusion and permit noninvasive detection of reperfusion within 30 minutes after release of an occlusive coronary arterial ligature. However, analysis of MM CK isoforms in plasma from patients has been hampered by the lack of availability of quantitative as opposed to qualitative methods. This study was performed to develop and validate an assay with the sensitivity and specificity needed for accurate quantification of MM CK isoforms in samples of plasma from patients. A rapid assay procedure will be required ultimately for prospective, clinical use. However, as a first step and for use in development and standardization of rapid assays, a procedure is needed for accurate qualification of isoforms even if its implementation is laborious and slow. The isoform composition of normal plasma was found to comprise 32.0% MMA, 34.9% MMB, and 32.7% MMC.(ABSTRACT TRUNCATED AT 250 WORDS)
Catheterization and Cardiovascular Diagnosis 13(1):26-32.
