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We measured levels of creatine kinase and its three isoenzymes in serum and blister fluid from 16 healthy volunteers. The BB-isoenzyme was found to be the predominant form in blister fluid while only the MM isoenzyme was found in serum. The levels of BB-isoenzyme in blister fluid decreased as the blisters aged. The source of BB-isoenzyme in blister fluid is most probably the damaged epidermis.
After eating a meal of poultry a 41-year-old man fell ill with severe diarrhoea, persistent high fever of around 39 degrees C, dehydration and somnolence. On admission to hospital physical examination was normal except for signs of dehydration. The blood count showed a leukocytosis (13,300/microliters) with 60% stab-form neutrophils. The erythrocyte sedimentation rate was raised to 49/82 mm. Also increased were the serum concentrations of creatinine (3.5 mg/dl), creatine kinase (179 U/l), lactate dehydrogenase (298 U/l) and C-reactive protein (16.8 mg/dl). Bacteriological and virological examinations of blood and stool were negative. A normal fluid and electrolyte balance was re-established. But as there was no improvement, ampicillin was administered, 2 g three times daily, then ciprofloxacin, 500 mg two times daily, and finally combined with metronidazole, 500 mg three times daily. Despite this treatment a chest radiograph on the tenth day revealed an infiltration in the left basal lung segment, and the legionella titre became positive at 1:2045. The antibiotic treatment was changed to 150 mg roxithromycin two times daily. The fever fell within 3 days and the diarrhoea stopped after 5 days. He was discharged free of symptoms after 24 days.
The mitochondrial isoenzymes of creatine kinase (MtCK), ubiquitous uMtCK and sarcomeric sMtCK, are key enzymes of oxidative cellular energy metabolism and play an important role in human health and disease. Very little is known about uMtCK in general, or about sMtCK of human origin. Here we have heterologously expressed and purified both human MtCK isoenzymes to perform a biochemical, kinetic and structural characterization. Both isoenzymes occurred as octamers, which can dissociate into dimers. Distinct Stokes' radii of uMtCK and sMtCK in solution were indicative for conformational differences between these equally sized proteins. Both human MtCKs formed 2D-crystals on cardiolipin layers, which revealed further subtle differences in octamer structure and stability. Octameric human sMtCK displayed p4 symmetry with lattice parameters of 145 A, indicating a 'flattening' of the octamer on the phospholipid layer. pH optima and enzyme kinetic constants of the two human isoenzymes were significantly different. A pronounced substrate binding synergism (Kd > Km) was observed for all substrates, but was most pronounced in the forward reaction (PCr production) of uMtCK and led to a significantly lower Km for creatine (1.01 mM) and ATP (0.11 mM) as compared to sMtCK (creatine, 7.31 mM; ATP, 0.68 mM).
No CK MM was found in human brain tissue. Both human and rat brain tissue, however, contain a non MM, non BB CK isozyme. The protein is membrane bound. Evidence is presented that it is a mitochondrial variant of the enzyme. This mitochondrial brain CK occurs in two forms. Both a low molecular mass form (Mr = 65,000) and a high molecular mass form are detectable. The isoelectric point of the low molecular mass form, Br-CKm1, is similar to the isoelectric point of CK MM. Complications may arise when techniques are used that try to separate the mitochondrial CK forms from CK MM on basis of differences in net change. The two mitochondrial brain CK forms are called Br-CKm1 and Br-CKm2. Whether these two enzymes are identical to the mitochondrial heart CK forms remains unclear.
The creatine kinase (CK) activity in human skeletal and cardiac muscle submitted to QAE-Sephadex chromatography was found to distribute into three peaks (m and h I, II, III fractions). Characterization according to electrophoretic behaviour, approximate molecular weight, thermal stability and immunological properties unambiguously demonstrated the coincidence of fractions I and III with reference MM-CK and MB-CK isoenzymes, while both cardiac and muscular forms II proved to escape any assignment within the dimeric (M, B) model. A higher molecular weight than for reference CK and the absence of interaction with BB-antiserum resulted for both forms II, which on the contrary were found to diverge as for reactivity to MM-antiserum and stability characteristics. Quantitation of the CK forms, attempted on a limited number of samples, confirmed the expected relative levels of MM-CK and BB-CK and gave evidence for the presence of hII- or mII-CK amounting up to 30% of the total activity in cardiac and skeletal muscle, respectively. The results of the study both confirm a complexity greater than supposed for the CK system and point to the inadequacy of several of the commonly used analytical approaches to derive correct information.
Creatine kinase isoenzyme activities in extracts of plasma, skeletal muscle, heart and brain tissue of domestic fowls were separated by anion exchange chromatography and tissue specific distributions of the isoenzyme designated MM-CK, BB-CK1 and BB-CK2 were demonstrated. The muscle isoenzyme (MM-CK) was the predominant form in plasma (99 per cent) and its activity increased in response to an episode of acute heat stress.
1. An in vivo change from creatine kinase MM3 into MM2 and finally into MM1 was described earlier in sera of patients during a post-myocardial infarction period. The same change takes place in vitro. 2. Parallel to this change, a turn-over from the MB2 isozyme into the MB1 form can be detected in vitro. 3. Immediately after the mixing of various extracts from heart tissue and other muscles with a serum with low creatine kinase activity, only MM3 and MB2 can be detected. MM1, MM2 and MB1 are not present in muscle cells. From these three observations it can be concluded that the phenomenon of the three MM and two MB isozymes is postsynthetic (i.e. epigenetic). Furthermore evidence is presented that the change from MM3 into MM1 is brought about by a thermolabile substance. This factor transforms one of the two M2 chains, present in the MM3 form, into a M1 chain. This results in the MM2 isozyme. Later on the second M2 chain is also transformed, resulting in the MM1 isozyme. The same mechanism is proposed for the observed change in MB pattern.
The regulation of glycosphingolipid (GSL) synthesis in culture by fusion-competent (E63) myoblasts and fusion-defective (fu-1) cells was examined. Upon reaching confluency E63 cells fused to form multinucleated myotubes and demonstrated many characteristics of developing skeletal muscle including induction of creatine kinase activity and a shift in creatine kinase isozymes to the MM isoform. The fu-1 cells displayed none of these characteristics, despite the fact that both cells were cloned from the same parental myoblast line (rat L8). There was a transient increase in the synthesis of total neutral GSLs by E63 cells at the time of membrane fusion. In contrast, neutral GSL synthesis by fu-1 cells gradually decreased with time in culture. The major GSLs synthesized by both cell types were lactosylceramide and ganglioside GM3, with more complex structures being observed with prolonged time in culture. Several glycosyltransferase activities were assayed at varying times in culture. Generally, the changes in activities fell into three groups. One group was maximally activated at the end of the culture period (GalT-3, GalNAcT-1 and GalT-6). Another group was maximally activated during the time of active membrane fusion (GlcT and SAT-1). A third group was maximally activated at the time of cell contact and the beginning of membrane fusion (GlcNAcT-1 and GalT-2). In terms of the times of maximal activation there were few differences between E63 and fu-1 cells, with one notable exception. The activity of GalT-2 (lactosylceramide synthase) in E63 cells increased dramatically upon contact and the beginning of membrane fusion, whereas there were no changes in GalT-2 activity in fu-1 cells during time in culture. These results support our hypothesis that membrane glycosphingolipids play an important role in the differentiation of skeletal muscle cells.
OBJECTIVE: To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro. METHODS: The skeletal muscle samples were obtained from 20 to 25-week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. RESULTS: The isolated myoblasts were spherical in cell suspension and spindle-like after attached to culture dishes. The myosin specialized immunohistochemical staining was strongly positive. A large quantity of skeletal muscle specialized creatine kinase (CK-MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. CONCLUSION: A large number of myoblasts can be available with digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection. The cultured myoblasts have good ability of proliferation and differentiation.
Mutant forms of DnaA protein, inert in a replication system composed of other purified proteins, are "activated" by DnaK and GrpE heat shock proteins (Hupp, T. R., and Kaguni, J. M. (1993) J. Biol. Chem. 268, 13137-13142). The effect of these heat shock proteins on DnaA5 and DnaA46 protein was separated from the event of DNA synthesis by incubation in two stages. Components necessary during the first stage for "activation" included GrpE and DnaK proteins, ATP at 0.2 mM or greater, and polyvinyl alcohol (8%) or glycerol, optimal at concentrations between 20 and 30%. An ATP regenerating system provided by creatine kinase and creatine phosphate was stimulatory. Addition of the activated form of DnaA5 or DnaA46 protein to a reconstituted system containing other purified replication proteins during the second stage of incubation resulted in DNA replication. Activation of DnaA5 or DnaA46 protein by heat shock proteins was thermolabile, suggesting that the temperature sensitivity of dnaA5 and dnaA46 mutants is related to this thermolabile interaction. A third heat shock protein, DnaJ protein, interfered with the activation of DnaA5 protein if present during the first stage of incubation. This inhibitory effect was less striking if included during the second stage of incubation. These results suggest a mechanism for regulation of the activity of DnaA protein.
We could detect skeletal muscle injury early after an acute exercise bout by measuring creatine kinase (CK, EC 2.7.3.2) MM isoforms in serum. Eleven men performed 120 alternating-arm, eccentric (muscle lengthening) biceps contractions with the intensity of each contraction being 110% of maximal concentric strength--a form of exercise previously shown to cause significant increases of CK in serum at 24 h and muscle soreness 48 h after exercise. Total CK and CK-MM isoform activities in serum were determined before and at 0.5, 0.75, 1, 1.5, 2, and 6 h after exercise. Using thin-film agarose gels and a rapid isoelectric focusing technique, we separated the MM isoforms into MM3 (skeletal muscle form), MM2, and MM1 (in vivo conversion forms). The isoforms reflected the MM form released into the serum from tissue as well as the conversion of one form to another. There were no significant increases in total CK from before to 6 h after exercise: 75 (SD 36) vs 91 (SD 33) U/L. However, CK MM3 in serum increased significantly (P less than 0.01) within 2 h after exercise from 22 (SD 6)% to 28 (SD 6)%. The MM3 to MM1 ratio also increased significantly (P less than 0.05) during this time, from 0.6 (SD 0.3) to 0.9 (SD 0.4). Thus, quantification of CK MM isoforms permitted very early detection of skeletal muscle enzyme release.
Rabbit heart mitochondrial creatine kinase has been recently shown to exist in two oligomeric forms: a dimer and an octamer, the latter being the form associated with the inner mitochondrial membrane [(1988) Biochem.Biophys. Res. Commun. 153,1310.]. We report here on the determination of the isoelectric points (pI) of the two purified forms by thin layer isoelectric focusing. The pI of the dimer is 8.2 and that of the octamer is 8.8; the former is higher by more than one pH unit than that of the cytoplasmic form MM-CK. It is proposed that the higher pI of the octamer is responsible for its binding to the inner membrane.
We describe two cases, hospitalized patients, in whom the activity of creatine kinase (EC 2.7.3.2) isoenzyme-MB was above normal. Both are particularly noteworthy in that creatine kinase-BB and a macro creatine kinase form thought to be type II of mitochondrial origin were also present. The macro creatine kinase component in both cases co-migrated electrophoretically with creatine kinase-MM but was easily identified after the latter was removed by precipitation with M-subunit-specific antibodies. In the first case, the patient had a readily diagnosable acute myocardial infarction while under observation in the cardiac intensive care unit: electrocardiographic changes and the rapid increase and decrease in total creatine kinase were as would be expected. In marked contrast, in the second case, we saw no abrupt changes in either of these characteristics. The latter patient's primary disease was a rectal carcinoma with massive metastases to the liver; however, the presence of abnormally high creatine kinase-MB activity raised the question of possible myocardial infarction.
Effect of oxidized and reduced forms of pyridine nucleotides on activity of creatine kinase BB was studied in rat brain. NAD did not affect the enzymatic activity at concentrations of 1.0-4.0 mM, while in presence of NADH activity of creatine kinase BB was gradually decreased as the coenzyme concentration increased. The plot of 1/V against 1/S at various NADH concentrations produced a series of lines intersecting at one point (-1/Km), thus indicating the noncompetitive type of brain cytoplasmic creatine kinase inhibition by reduced pyridine nucleotides. Apparent Ki value for NADH was about 1.5 mM which was lower than the known parameter for cytoplasmic creatine kinase isozymes from skeletal and heart muscles. Dependence of brain creatine kinase BB on the rate of pyridine nucleotide reduction suggested a possible mechanism for the enzyme regulation under hypoxic conditions.
The apparent activation energy of the CK reaction as well as the Michaelis-Menten constants and the isoelectric point of CK MM can be used as indices for the mean age of the CK M-chain in the blood in vivo and in vitro. Modifications in the CK M-chain take place in vivo in the blood and in vitro in a serum matrix. Gradual increases in the apparent activation energy are also observed both in vivo and in vitro. It is confirmed that the modification in the CK M-chain causes a rise in the apparent activation energy, mu. A gradual increase in apparent activation energy, due to the ageing process of the CK M-chain, was observed after myocardial infarction. A significantly increased value for u was observed at the time that total CK activity already had returned to reference values. In spite of the normal CK value, the apparent activation energy still indicated that there had been myocardial damage. The Michaelis-Menten constants for creatine phosphate and ADP are also influenced significantly by the modification in the M-chain. While the apparent activation energy increases, the Michaelis constants decrease in the order MM3, MM2, MM1. The Michaelis-Menten constants for both ADP and CrP can be used as an index for the mean age of the enzyme in the blood. The Michaelis-Menten constants for CrP and ADP show significant variations with the measuring temperature for virtually all CK MM forms.
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.
The MM and MB isoenzymes of creatine kinase exist in serum as a collection of at least three major MM and two major MB isoforms. Each of these are derived from single tissue MM and MB isoforms, which are converted to these other forms by carboxypeptidase N after their release from necrotic skeletal and myocardial tissue. Measurement of the MM isoforms in ischemic heart disease is useful for early diagnosis of acute myocardial infarction and for the noninvasive determination of coronary artery reperfusion for infarction patients receiving thrombolytic therapy. Because MM is also released in acute skeletal-muscle disease, MB isoform measurements may have the highest clinical sensitivity. These determinations are important for providing objective information to cardiologists who need to make critical decisions concerning the management of these patients. I review the procedures for treating patients with myocardial infarction, the potential role of CK isoforms, and the methods currently available for isoform analysis, including high-resolution electrophoresis, isoelectric and chromatofocusing, and liquid chromatography. Rapid and highly sensitive methods are needed for implementation of CK-MM and MB isoforms for prospective emergency determinations for patients with acute myocardial infarction.
The case of a patient with bronchial neoplasm is described. A non-typical form of creatine kinase found in the plasma gave rise to a false positive CK MB test by the immunoinhibition procedure. Electrophoresis on agarose gels beside a normal MM band showed an additional band between the MB and the MM band of a standard mixture. It could be shown by an immunoprecipitation test using precipitating antibodies against CK-MM and CK-BB that the creatine kinase of the abnormal band consists entirely of the MM-type, which after treatment with polyethylene glycol not only became susceptible to inhibiting anti-M antibodies, but also showed the electrophoretic motility of CK-MM. Gel filtration chromatography clearly demonstrated the macromolecular nature of this abnormal creatine kinase.