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Creatine kinase isoenzymes (MM, MB and BB) were isolated from human tissue by ion-exchange chromatography. The B subunit was found to be more heat sensitive than the M subunit. BB and MB isoenzymes respond similarly to heat inactivation. Our results are in contrast with the body temperature inactivation of the brain isoenzyme reported by Lindsey and Diamond.
Properties of human creatine kinase isoenzymes (MM, MB and BB) are investigated. The most pronounced differences in properties of these isoenzymes are found under their urea inactivation, heat denaturation and the inhibition by rabbit antisera to isoenzymes. Differences in values of the Mikhaelis constant and substrate and pH dependencies are much less pronounced. The presence of ADP stabilizes creatine kinase isoenzymes under conditions of urea and heat inactivation. Properties of hybrid MB isoenzymes are found to be intermediate with respect to MM and BB isoenzymes. A mode of the interaction of M and B subunits in dimeric molecules of creatine kinase isoenzymes is discussed.
We measured creatine kinase (EC 2.7.3.2) activity in 1009 serum samples from 538 patients in the intensive-care units of the University of Texas Medical Branch hospitals. Creatine kinase isoenzymes migrating cathodal to skeletal muscle creatine kinase (CK-MM) on cellulose acetate electrophoresis were found in sera from 14 of the 538 patients. Creatine kinase, lactate dehydrogenase (EC 1.1.1.27), aspartate aminotransferase (EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2) activities were abnormally increased in these 14 patients. Liver lactate dehydrogenase isoenzyme (LDH5) and cardiac creatine kinase isoenzyme (CK-MB) were abnormally increased in 12 and eight of these patients, respectively. Ten of the 14 patients died during their hospital admission. We believe the creatine kinase isoenzymes that migrated cathodal to skeletal muscle creatine kinase (CK-MM) were of mitochondrial origin.
12 cases are described, which show high serum creatine kinase-BB levels, as well as atypical creatine kinase activity band located between normally migrating creatine kinase-MM and creatine kinase-MB. It is shown that the altered properties of the serum creatine kinase-BB, namely its molecular size, heat resistance, electrophoretic mobility, but not its immunological behavior, are caused by complexing with kappa-chains of immunoglobulins G or A. The complex occurring in vivo could also be produced in vitro by using purified patients' IgG and human creatine kinase-BB.
The brain isoenzyme of creatine kinase (CK) (EC 2.7.3.2) has been identified in the serum of 21 of 950 patients tested. Samples were received over a period of about 20 months. Analysis was performed by electrophoresis on agarose gel with isoenzyme bands visualized by fluorescence after enzyme coupled reactions producing NADH. Specialized substrate preparations were used to rule out the possibility that observed fluorescence was due to enzymatic activity other than that of CK isoenzymes. Patients who were hypothermic and/or had undergone cardiopulmonary resuscitation accounted for 19 of the 21 patients showing the brain isoenzyme. The inciting mechanism for the release of CK1, into the circulation is not clearly understood.
The MB isoenzyme of creatine kinase (CK) may be prepared in vitro from rabbit serum containing only the MM and BB isoenzymes, by means of a hybridization technique. The MM and BB dimers dissociate in 4 mol/L urea, which allows random recombination of M and B monomers. A liquid CK-isoenzyme control can be made from mixtures of rabbit sera obtained after hybridization and stabilized with glycerol and 25 mmol of 2-mercaptoethanol per liter. A liquid control stored at 4 degrees C showed good stability over a three-month period, declining to a mean residual activity of CK of approximately 90% after three weeks and a mean residual activity of MM, MB, and BB of 80--85% after six weeks. At 25 degrees C, CK activity of the liquid control declined to 75--80% after the fourth week. CK-BB at 25 degrees C was the least stable isoenzyme, declining to 75% after the third week and reaching 60% of activity after 12 weeks. CK-MB and CK-MM showed approximately 10--15% less stability at 25 degrees C than at 4 degrees C.
Serum MB isoenzyme of creatine kinase is the most specific and sensitive indicator of myocardial infarction. Despite these virtues, its clinical use is handicapped by rapid normalization after the onset of myocardial injury. In patients suffering brain accidents, the increase of serum CK-MB isoenzymes indicates stimultaneous myocardial damage and means poor prognosis.
We describe a sensitive, specific radioimmunoassay for the BB isoenzyme of creatine kinase (CK-BB) in serum. A sequential saturation assay was used to achieve sufficient sensitivity to detect the isoenzyme in 100-microliter serum samples of all healthy persons and patients tested. Bound and free antigen were separated by a second antibody system. Large excesses of purified isoenzyme MM did not react in the assay. Cross reactivity of two preparations of CK-MB was only 1 to 7+. The 95th percentile of serum CK-BB in 208 healthy adults was 6.2 microgram/liter. Within-assay and between-assay precision ranged from 5.5 to 11.9% and 9.7 to 13.6%, respectively.
Chicken heart muscle contains almost exclusively the BB isoenzyme of creatine kinase (CK), its myofibrils, moreover, lack an M-line. This tissue thus provides an interesting contrast to skeletal muscle, in which some of the MM-CK present as predominant CK isoenzyme is bound at the myofibrillar M-line. Approx. 2% of the total CK activity in a chicken heart homogenate remains bound to the myofibrillar fraction after repeated washing cycles; both the fraction and the absolute amount of CK bound are about threefold lower than in skeletal muscle. Almost all of the bound enzyme is located within the Z-line region of each sarcomere, as revealed by indirect fluorescent-antibody staining with antiserum against purified chicken BB-CK. After incubation with exogenous purified MM-CK, positive immunofluorescent staining for M-type CK at the H-region of heart myofibrils was observed, along with weaker fluorescence in the Z-line region. Chicken heart myofibrils may thus possess binding sites for both M and B forms of CK.
Activity of creatine kinase isoenzyme MB was maintained for 5 days at 4 degrees C in 5 ml whole blood collected in specimen bottles containing 9.5 mg EGTA and 2 microliters beta-mercaptoethanol for glutathione-activated CK-MB assays and 7.5 mg EDTA for N-acetyl-L-cysteine-activated CK-MB assays.
A method is developed for detection and quantitative determination of the creatine kinase isoenzymes during electrophoresis in agar gel. They are found in the agar gel block by formation of fluorescent sites due to combination of the isoenzymes reaction product: creatine with ninhydride in the alkaline medium followed by fluorophore quantitative elution. The method is specific, simple, highly sensitive. It was used to study mobility of creatine kinase of mitochondria and isoenzymes of the rat myocardium mytochondria sarcoplasmic fraction during electrophoresis under different conditions. It is established that mobility of creatine kinase of mitochondria differs from that of isoenzymes. During electrophoresis in agar gel, contrary to polyacrylamide gel, it arranges relative to sarcoplasma isoenzymes depending on the buffer used.
During the first 2 days of culture of myogenic chicken cells only minute amounts of mRNA for a subunit of creatine kinase, M-CK, could be found in cultures from the 3rd and 4th day; however, a dramatic increase of mRNA for the muscle-specific M-CK was observed. The mRNA for B-CK, another subunit of creatine kinase, was readily detectable in young cultures increased up to the 3rd day in culture but decreased significantly 1 day later. The comparison of the ratios of mRNA concentrations for M-CK and B-CK in polysomal and total cellular RNA did not reveal significant differences in polysomal and total cellular RNA from 24-, 48-, and 68-h cultures, indicating that specific accumulation of a nonpolysomal form containing translatable mRNA for M-CK prior to its appearance in the polysomes does not seem to represent a major mechanism of regulation. In RNA from cells grown in 5-bromo-2'-deoxyuridine (BrdUrd) for 3 days and subcultured into the same medium for another 3 days only mRNA for B-CK was found. If cells, however, were subcultured into standard medium both mRNAs, the one for B-CK and the one for M-CK could be detected. BrdUrd inhibition seems to affect only the mRNA for M-CK and not the one for B-CK. Removal of BrdUrd from the cultures probably allows new transcription or processing of mRNA for M-CK, or both.
Measurement of the mass concentration of serum enzymes by radioimmunoassay provides direct quantitation of specific isoenzymes and may be less subject to some of the limitations of traditional assay procedures for enzymes. We describe the development of a sensitive and specific radioimmunoassay for the muscle isoenzyme of creatine kinase, CM-MM, in human serum. CK-MM, purified from human skeletal muscle, was used to raise high-titer antisera and for iodination by the Chloramine T method. The radioimmunoassay required 50 microliter of sample, utilized a double-antibody separation method, and was completed in 24 h. Cross reactivity with CK-BB was virtually zero, 3--17% with CK-MB. The mass concentration of CK-MM in the serum of healthy subjects ranged from 36 to 1668 microgram/liter and correlated closely with total CK enzymatic activity. Serum concentrations of CK-MM from casually selected patients correlated less well with total CK enzymatic activity, suggesting the existence of other CK isoenzymes or the presence of inactive forms.
A method for determination of the creatine kinase isoenzyme MB (CK-MB) is reported: separation of the isoenzymes was done by electrophoresis and the activity of the isoenzyme bands quantitated by scanning fluorometry. Total CK activity was used for calculation of CK-MB level. The precision of the method was satisfactory: coefficient of variation 5-10%. Its accuracy good: CK-MB was consistently found in high concentrations in tissue extracts of myocardium, but was virtually absent in skeletal muscle and could not be demonstrated in serum from patients with skeletal muscle damage. The sensitivity of the method fitted its clinical use: CK-MB was undetectable (less than 5 U/l) in normal sera, below 30 U/l in seventy-six out of seventy-seven patients in whom the diagnosis of acute myocardial infarction (AMI) was disproved, and above 30 U/l in all seventy-two patients with AMI according to WHO criteria. The CK-MB concentration in serum rises to a maximum about 20 h after onset of clinical symptoms of AMI and reaches baseline levels 20-30 h later. The electrophoretic CK-MB method is easy, fast and reliable and is considered as an important diagnostic test for AMI.
The activity of creatine kinase isoenzyme BB (CK-BB) was determined in serum of healthy adults and in patients undergoing maintenance hemodialysis and with kidney transplant. In the healthy adults examined, an activity of 0.56 +/- 0.16 U/l (mean +/- SD) was found. The arithmetic mean of CK-BB activity in patients with renal insufficiency under hemodialysis was 1.42 +/- 0.87 U/l and differed from that of the healthy group. The CK-BB activity in patients with kidney transplant was not different from that of the control group. The occurrence of CK-BB in serum is discussed from diagnostic and methodological point of view.
We compared (a) the frequency of detection of isoenzyme MB of creatine kinase (CK; EC 2.7.3.2) in serum of patients undergoing coronary-artery bypass surgery, (b) the interval during uhich its activity was supranormal in serum, and (c) an index of the amount of CK released into blood ("CK-MB area") with postoperative electrocardiographic changes in 80 patients. The frequency of detection of CK-MB is a function of frequency of sampling during the early postoperative period. Because the duration of appearance and the calculated CK-MB area increased as the electrocardiogram became more specific for infarction (p less than 0.01), a twice-daily sampling schedule proved clinically relevant. Only 5.4% of patients had electrocardiographic evidence of infarction when CK-MB was absent by the second postoperative morning. When CK-MB was still detected at that time, 69.6% of patients had persistent new Q waves, consistent with infarction. In three patients who died postoperatively, significant myocardial necrosis was demonstrated. All three had had persistently increased values for CK-MB, related to electrocardiographic changes of infarction in one patient and ischemic changes in two. Evidently CK-MB is a more sensitive indicator of myocardial necrosis than the electrocardiogram and CK-MB area should be a useful criterion in evaluating methods of intra-operative myocardial protection.
In this investigation we have shown that severe liver disease with cerebral involvement can be followed by the presence of BB creatine kinase isoenzyme in serum. Groups of rats were injected with a bile acid mixture, oleic acid, albumin and normal saline respectively. Bile acids or/and oleic acid induced BB isoenzyme to leak from central nervous tissue and this can be measured in serum. Albumin binding prevented this leakage. The experiments support our hypothesis, based on biochemical findings in human liver failure, that detergent and surface activity properties of the above mentioned components are able to produce this specific brain isoenzyme leakage.
DEAE-cellulose chromatography (pH 7.0) of human heart extracts revealed the presence of three creatine kinase isoenzymes. The CK3 (skeletal muscle) isoenzyme was not retained on the column under these conditions. The CK2 (heart) and CK1 (brain) isoenzymes eluted at a conductivity of 5.5 +/- 0.6 m omega-1 and 11.4 +/- 1.2m omega-1, respectively. When DEAE-cellulose chromatography was performed at pH 8.0, CK2 eluted at a slightly higher conductivity, 6.5 m omega-1, whereas CK1 eluted as before 12.0 m omega-1. DEAE-cellulose chromatography of CK2 and CK1 isoenzymes in the presence of serum protein, and serum albumin had no significant effect on the elution of CK2 at pH 7.0 and 7.4, and on the elution of CK1 at pH 7.0 and 8.0 However, serum and serum albumin decreased the affinity of CK2 for DEAE-celluose at pH 8.0, and caused this isoenzyme to elute at a conductivity of 3.0-3.5 m omega-1. The decreased affinity of CK2 for DEAE-cellulose was not due to aggregation of CK2 with albumin or some other serum protein, but was related to the amount of albumin applied to the column.