[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] ABSTRACT: Because interactions between platelets and plasminogen activators are likely to influence the clinical outcome of coronary thrombolysis, we characterised changes in aggregation of platelets from canine and human whole blood exposed to pharmacologic concentrations of tissue-type plasminogen activator (t-PA) or streptokinase (SK). Aggregation of canine platelets in response to ADP or to collagen decreased markedly after exposure to 5000 ng/ml of t-PA. In contrast, canine platelets exposed to 250 U/ml of SK exhibited little change or slight enhancement of aggregation. After exposure of human platelets to 5000 ng/ml of t-PA, aggregation was decreased significantly in response to ADP and to collagen. In contrast to canine platelets, aggregation of human platelets exposed to 250 U/ml of SK was markedly decreased. The diminished aggregatory response of human platelets exposed to t-PA or SK was both time- and concentration-dependent. It appeared to be dependent on generation of active plasmin as evidenced by depletion of α2-antiplasmin and plasminogen and by preservation of normal aggregation when platelets were exposed to either plasminogen activator and aprotinin concomitantly. The disparate effects of SK on aggregation of canine and human platelets appeared to be related to the more modest activation of plasminogen by SK in canine compared with human blood. Results of surface radiolabelling and immunoblotting experiments indicated that exposure of human platelets to t-PA led to diminution of membrane associated fibrinogen. Thus, exposure of platelets to pharmacologic concentrations of plasminogen activators appears to influence platelet function by a plasmin-dependent mechanism resulting in reduction of membrane associated fibrinogen.
[Show abstract][Hide abstract] ABSTRACT: Conventional plasma isoenzyme and enzyme values usually are normal during the first few hours of acute myocardial infarction. Thus definitive diagnosis may be delayed. We have shown recently that infarction in dogs can be detected within 1 hr after coronary occlusion by analysis of relative activities of MM creatine kinase (CK) isoforms in plasma. Isoforms of MM CK evolve through posttranslational modifications in plasma of the form released from tissue (MMA) to MMB and MMC. In this study we quantified changes in isoform profiles in the first available plasma samples from patients with evolving myocardial infarction, from patients with angina, and from normal subjects. In the 26 control subjects, the ratio of MMA to MMC was 1.09 +/- 0.4 (SE) (range 0.31 to 3.1; upper limit of normal [defined as the mean plus 2 SD] 2.5). In the seven control patients with coronary artery disease, the ratio of MMA to MMC was 1.3 +/- 0.3 with a range of 0.5 to 2.5. In contrast, among the 28 patients with acute myocardial infarction, the ratio of MMA to MMC in the first available plasma sample averaged 14.6 +/- 4.5 (p less than .01 compared with both control groups). First available samples were obtained 3.9 +/- 0.4 hr after the onset of pain. In 24 of 28 patients (86%) the ratio of MMA to MMC was greater than 2.5.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: Creatine kinase (CK; EC 184.108.40.206) plays an important role in energy metabolism in brain and muscle. Expression of CK isoenzymes is regulated during development and is tissue specific. To define the structures of canine CK isoenzymes and to elucidate the mechanism of regulation in their expression, CK cDNA clones from dog myocardium were isolated. Myocardial CK mRNA is predicted to encode a protein of 381 amino acids. The nontranslated regions of the mRNA comprise at least 38 bases at the 5' end and exactly 345 bases before the poly(A) tail. Partial protein sequences of dog muscle (M) CK and brain (B) CK subunits were determined and compared with the derived amino acid sequence of the myocardial enzyme and of M CK subunits of other species. The M CK subunits from different species share a very high degree (83-96%) of sequence identity. Dog M and B subunits share extensive sequence identity (74%), a degree of similarity not previously suspected. Southern blot analysis suggests that a CK gene family exists. These observations imply that evolutionary changes in the M CK subunit structure are constrained by the need for preservation of functional properties other than the kinase activity. This conservation is consistent with the possibility that the M subunit plays a structural role in cardiac and skeletal muscle.
Proceedings of the National Academy of Sciences 01/1986; 82(24):8394-8. DOI:10.1073/pnas.82.24.8394 · 9.81 Impact Factor
[Show abstract][Hide abstract] 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. · 4.57 Impact Factor
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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. · 2.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Improved, and relatively simple procedures are described for the purification of human and canine creatine kinase isoenzymes with much higher yields (30 to 60%) and purity (450 kU/g) than previously possible. Contaminant albumin was removed from the MB preparation by albumin antibodies attached to Sepharose-4B. All isoenzyme preparations exhibited a single protein band on SDS gel electrophoresis. Using specific antisera to the isoenzymes and albumin the preparations were shown to be immunochemically pure and to give reliable results in a radioimmunoassay.
[Show abstract][Hide abstract] 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.