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PubMed1977

Measurement of creatine kinase Z in human sera using a DEAE-cellulose mini-column method.

A DEAE-cellulose mini-column method has been developed which allows for the quantitation in human serum of creatine kinase Z, a sub-band of creatine kinase first described by Lim ((1975) Clin. Chem. 21, 975, Abstract 181) and Sax et al. (Sax, S.M., Moore, J.J., Giegel, J.L. and Welsh, M. (1976) Clin. Chem. 22, 87). We have shown that creatine kinase Z is rather unstable in nature, and converts to a form which electrophoreses with creatine kinase MM on agarose gel electrophoresis. CK-Z is not present in normal human serum. CK-Z is present in human heart extracts, in patients with myocardial infarcts and in patients with skeletal muscle trauma. In infarct patients CK-Z levels paralleled changes in the CK-MB levels. CK-Z ranged in activity from 8.8-67.2 I.U. whereas CK-MB ranged from 29.6-121.6 I.U. in infarct patients. CK-Z and CK-MB activity were measured at or close to the peak rise in total CK activity.

Brain↗

PubMedDate not supplied

False elevation of amniotic fluid enzymes of relevance for prenatal diagnosis: creatine kinase measurement in relation to Duchenne muscular dystrophy.

The creatine kinase activity of amniotic fluid was measured in samples collected at fetoscopy. In our first study, the control sample range was 0.25 IU/l, although four samples had activities of 35-85 IU/l. Elevated values did not correlate with the activities in the fetal or maternal circulations. Electrophoresis revealed the presence of the BB isozyme of creatine kinase rather than just the MM form as expected. This suggested that the source of the elevated enzyme activity was from the myometrium, damaged by insertion of the trocar and cannula. In a further series the first 2 ml of amniotic fluid withdrawn yielded a much higher creatine kinase activity than a second aliquot. A control series of such second samples (first 2 ml discarded) gave an activity range of 0-7 IU/l with no spuriously high values. This compares favourably with a series from single samplings taken by amniocentesis. Normal creatine kinase activities were found in the amniotic fluids from 20 pregnancies at risk of Duchenne muscular dystrophy. We conclude that for accurate measurement of amniotic fluid enzyme activity the first portion withdrawn should be discarded. Amniotic fluid creatine kinase activity is of no value for the prenatal diagnosis of Duchenne muscular dystrophy.

Amniocentesis↗

PubMed1981

Induction by butyrate of differentiated properties in cloned murine rhabdomyosarcoma cells.

A cell line derived from the murine rhabdomyosarcoma BW10139 (Dexter, Cancer Res. 37: 3136, 1977) was subcloned and examined with respect to growth and myogenic characteristics in the presence and absence of 1 mM butyrate. Without butyrate, these cells behave as typical transformed cells: they grow rapidly and chaotically, do not form multinucleated muscle fibers and have little or no creatine kinase activity. In the presence of 1 mM sodium butyrate or butyric acid, growth slows, cells become arranged in whorl patterns, and creatine kinase activities increase to levels comparable to those found in normal chick myoblasts immediately prior to cell fusion. The increase in creatine kinase activity is detectable within 2 h exposure to butyrate, reaches a maximum by 24 h, and the elevated level can be maintained for at least six weeks. The induction is reversible upon sequential addition, deletion, and readdition of butyrate to the culture medium. Isoenzyme analyses demonstrated that only the BB form of creatine kinase is induced; MM creatine kinase was not detected. Although formation of multinucleated cells increases after exposure to butyrate, no typical myotubes form. The results suggest that this rhabdomyosarcoma cell line can, under appropriate conditions, re-express some properties characteristic of skeletal muscle, but not the complete muscle phenotype.

Animals↗

PubMed1983

Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes.

The nonreceptor, peripheral v proteins (Mr 43,000 proteins) are conspicuous components of the acetylcholine receptor-rich membranes and the Torpedo electrocyte, so far devoid of any known enzymatic function. Creatine kinase (adenosine 5'-triphosphate:creatine N-phosphotransferase, EC 2.7.3.2) is identified in distinct polypeptides belonging to the family of v proteins. Embryonic (70- to 90-mm embryos), neonatal, and adult electric organs of Torpedo marmorata contain two isoenzymes of creatine kinase: the BB (brain) and the MM (muscle) forms. The proportion of the two isoenzymes does not appear to change in the course of ontogenic and postnatal development. Only the BB isoenzyme appears to be associated with the acetylcholine-rich membranes in adult Torpedo. The creatine kinase can be purified to homogeneity by chromatographic procedures that exploit the richness in free sulfhydryl groups of the enzyme. Specific activities of 150 units/mg are obtained from electric tissue. The enzyme subunits identified by two-dimensional gel electrophoresis and immunoblotting techniques have pI values in the 6.0-6.5 region and apparent molecular weights in the 40,000-43,000 range, the latter values depending on redox conditions.

Animals↗

PubMed1984

Reprogramming cell differentiation in the absence of DNA synthesis.

We examined whether the activation of muscle gene expression in nonmuscle cells required DNA synthesis. Human fibroblasts from amniotic fluid and fetal lung were fused with differentiated mouse muscle cells in the presence or absence of the DNA synthesis inhibitor, cytosine arabinoside. In the stable heterokaryons formed, the human contractile enzyme, MM-creatine kinase (CK), and the cell surface antigen, 5.1H11, were detected in comparable amounts regardless of whether DNA synthesis had occurred. A single cell analysis revealed that the efficiency of gene activation was high and that DNA synthetic activity was not affected by the ratio of muscle to nonmuscle nuclei in the heterokaryons. In addition, muscle gene expression was not restricted to the G1 phase of the cell cycle. We conclude that cell differentiation can be reprogrammed in heterokaryons regardless of cell cycle phase and in the absence of detectable DNA synthesis.

Animals↗

PubMed1983

Mitochondrial creatine kinase from human heart muscle: purification and characterization of the crystallized isoenzyme.

An isoenzyme of creatine kinase [ATP: creatine N-phosphotransferase, EC 2.7.3.2] was isolated in pure, crystalline form from mitochondria of human heart muscle. The enzyme has a molecular weight of 84,000 and consists of two subunits with identical molecular weight, each containing two reactive sulfhydryl groups. The enzyme has a specific activity of 15 +/- 2 U/mg in the direction of creatine phosphate synthesis at optimum pH of 8.7 and of 45 +/- 5 U/mg in the direction of ATP synthesis at optimum pH of 6.7. Mitochondrial creatine kinase has Michaelis constants of 1.70 mM for MgATP2-, 8.00 mM for creatine, 0.15 mM for MgADP-, and 3.00 mM for creatine phosphate. The mitochondrial enzyme differs from the other creatine kinase isoenzymes, i.e. from muscle (CK-MM), brain (CK-BB), and their hybrid form (CK-MB) in (I) its amino acid composition and its amino terminal amino acid sequence, (II) its electrophoretic mobility, and (III) its immunological properties. It thus constitutes a fourth isoenzyme of creatine kinase. By analogy to CK-MM, CK-BB, and CK-MB, the mitochondrial isoenzyme was designated as CK-MiMi.

Amino Acid Sequence↗

PubMed2006

CK-MM autoantibodies: prevalence, immune complexes, and effect on CK clearance.

Although the blood level of creatine kinase (CK) is the most commonly used marker of muscle injury, there is marked interindividual variability in this measure. Part of this variability may be attributed to variability in the rate of CK clearance from the circulation. In this study, we assessed the possibility that CK-MM autoantibodies form immune complexes with CK following muscle injury and subsequently affect the CK clearance rate. Using an enzyme-linked immunosorbent assay, CK-MM autoantibodies were detected in all 25 human subjects studied but the levels varied greatly. Using protein A-sepharose, the percentage of the plasma CK activity found in immune complexes was determined to be correlated with the CK-MM autoantibody level at lower CK levels (<1,022 U/L). When CK-MM antibodies were administered to mice, plasma CK activity following a bolus CK injection was reduced by 11%-32%. We conclude that CK-MM autoantibodies can modulate the rate of CK clearance from the circulation. Thus, the relatively low blood CK levels seen in some individuals following injury may be attributed partly or entirely to an autoantibody-enhanced clearance of CK.

Adult↗

PubMed1996

Marked elevations of serum creatine kinase activity associated with antipsychotic drug treatment.

Serum creatine kinase (SCK) activity of the skeletal muscle (MM) form is sometimes moderately increased in acutely psychotic patients and may be massively increased as a result of muscle damage. The objective of this study was to characterize the SCK increases in patients treated with novel antipsychotic drugs. SCK activity and myoglobinuria, an index of gross muscle damage, were monitored at varying intervals in schizophrenic or schizoaffective patients treated with antipsychotic drugs. Possible causes of increases in SCK activity, such as trauma, excessive physical activity, exacerbation of psychosis, were assessed. Fifteen instances of massive increases in SCK activity were observed in 11 out of 121 patients (10%) treated with the following antipsychotic drugs: clozapine, loxapine, haloperidol, melperone, risperidone, or olanzapine. These increases in SCK activity were of the MM type and ranged from 1,206 to 177,363 IU/L (median, 9,600 IU/L). Thus, they were much larger than the increases usually found in acutely psychotic patients or patients with neuroleptic malignant syndrome (range, 500-3,000 IU/L). Only the patient with SCK activity of 177,363 IU/L had rhabdomyolysis as evidenced by myoglobinuria. The onset of the increases was from 5 days to 2 years after initiating treatment, and the increases lasted 4 to 28 days (median, 8 days). Flulike symptoms were present in two of the patients, but the others were asymptomatic. The increases were self-limiting in three cases, despite continuing treatment. Two of three cases rechallenged with the same drug again developed large increases in SCK activity within a week. It is unlikely these increases in SCK activity are related to acute psychosis, trauma, or the neuroleptic malignant syndrome. The increase in SCK activity may reflect the ability of the drugs to increase intermittently cell membrane permeability, especially in skeletal muscle, in some vulnerable subjects. A possible role of serotonin in this process is suggested by the pharmacology of most of the offending drugs. However, in some instances, the increases may have been unrelated to drug treatment. There was no evidence that these increases in SCK activity, despite their magnitude, compromised renal function. Routine monitoring of SCK activity of myoglobinuria during treatment with the antipsychotic drugs studied here is probably not necessary.

Adult↗

PubMed2003

Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state.

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.

Animals↗

PubMed1983

Investigation of the heterogeneity of the MM isozyme of creatine kinase.

MM creatine kinase (MM CK) from human skeletal muscle was resolved into 21 subspecies by isoelectric focusing. Rabbit and bovine MM CK's were also shown to have patterns of 21 bands. These results are compatible with the presence of six different M subunit forms in skeletal muscle, which by random association into dimers, could produce 21 different subspecies of creatine kinase. Two-dimensional isoelectric focusing of human MM CK yielded a diagonal pattern, indicating that the various enzyme subspecies were not induced by ampholyte-protein interaction. Heat inactivated serum appeared to stabilize various CK subspecies and also produced one further anodally migrating MM CK form. Fresh serum had combined inhibitory and stabilizing effects on the MM CK subforms, and allowed for the identification of four anodally migrating CK subspecies not present in tissue extract. These findings are of critical importance because of the widespread use of CK isoenzymes in serum for diagnostic purposes.

Animals↗

PubMed1989

Treatment of human muscle creatine kinase with glutaraldehyde preferentially increases the immunogenicity of the native conformation and permits production of high-affinity monoclonal antibodies which recognize two distinct surface epitopes.

Treatment of human muscle creatine kinase (MM-CK) with glutaraldehyde produced highly aggregated forms which retained the native antigenic structure. Immunization of BALB/c mice with CK aggregates instead of untreated CK produced over ten-fold higher titres of antibody against native CK without increasing the titres of antibody against denatured enzyme. Production of high-affinity monoclonal antibodies specific for both the muscle isoenzyme and the native conformation became possible where the use of untreated CK had failed. Four monoclonal antibodies have been characterized by an epitope mapping technique and compared with a commercially available monoclonal antibody. One antibody has a much higher affinity for MM-CK than the other three and the commercial antibody. Competition studies show that it also recognizes a different epitope on the CK surface from the other three monoclonal antibodies which bind to the same surface region as the commercial antibody. Immunoassays based on the high affinity antibody can easily measure less than 1 ng of CK, a sensitivity comparable to, or better than, standard enzymatic assays.

Antibodies, Monoclonal↗

PubMed1987

Fetal-type creatine kinase in rat fast and slow muscles during denervation and reinnervation.

Creatine kinase (CK) has three forms of isozymes; CK-BB, CK-MB, and CK-MM. In adult rats they show a specific tissue distribution: the BB form in the brain, the MB form in the heart, and the MM form in skeletal muscle. In embryonic skeletal muscles only the BB and MB forms are found. Adult slow-twitch muscles contain more fetal type creatine kinase (CK-B) than do fast-twitch muscles. In the present experiment the effect of denervation and reinnervation on the CK-B concentration was investigated in rat fast (extensor digitorum longus)- and slow (soleus)-twitch muscles by a highly sensitive immunoassay. Denervation of these muscles produced a progressive increase in CK-B concentration in both muscles. When the sciatic nerve was cut and immediately sutured, the CK-B concentration in both muscles showed a gradual reduction after an initial increase. By the 34th postoperative week the CK-B concentration in the soleus was about one-half that of the contralateral control, whereas that in the extensor digitorum longus was nearly normal. After cross union of the nerves innervating the muscles, the CK-B concentration in the soleus was reduced at 35 weeks to about one-half normal, but that in the extensor digitorum longus was always higher than the control value. After self-reunion of the nerves, the CK-B concentration at the 20th week was approximately normal in the extensor digitorum longus and significantly increased in the soleus. We suggest that the motoneurons normally innervating the extensor digitorum longus have a greater capability in suppressing the production of CK-B than do the soleus motoneurons.

Animals↗

PubMed2005

Characterization of creatine kinase isoforms in herring (Clupea harengus) skeletal muscle.

It is known that mitochondrial creatine kinase (MtCK) in mammals is always expressed in conjunction with one of the cytosolic forms of creatine kinase (CK), either muscle-type (MM-CK) or brain-type (BB-CK) in tissues of high, sudden energy demand. The two creatine kinase (CK) isoforms were detected in herring (Clupea harengus) skeletal muscle: cytosolic CK and mitochondrial CK (MtCK) that displayed the different electrophoretic mobility. These isoforms differ in molecular weight and some biochemical properties. Isolation and purification procedures allowed to obtain purified enzymes with specific activity of the 206 micromol/min/mg for cytosolic CK and 240 micromol/min/mg for MtCK. Native M(r)s of the cytosolic CK and MtCK determined by gel permeation chromatography were 86.000 and 345.000, respectively. The results indicate that one of isoforms found in herring skeletal muscle is a cytosolic dimer and the other one, is a mitochondrial octamer. Octamerization of MtCK is not an advanced feature and also exists in fish. These values correspond well with published values for MtCKs and cytosolic CK isoforms from higher vertebrate classes and even from lower invertebrates.

Animals↗

PubMed1994

Creatine kinase activity associated with the contractile proteins of the guinea-pig carotid artery.

Activity and role of creatine kinase associated with contractile proteins of vascular smooth muscle have been investigated using skinned guinea-pig carotid artery rings. Membrane solubilization was performed with the detergent Triton X-100. Creatine kinase activity, isoenzyme profile as well as mechanics were performed on the Triton skinned carotid artery rings. Total creatine kinase activity was 47.3 +/- 9.3 IU g-1 ww and electrophoresis showed BB, MB, and MM isoforms (BB-CK being the predominant isoenzyme). One hour incubation with Triton X-100, produced predominantly BB-CK remaining with the myofibrils with some MB, representing 23% of the preskinned creatine kinase activity. When relaxed carotid artery rings were exposed to pCa 9 in the presence of 250 microM ADP, 0 ATP, and 12 mM phosphocreatine, tension was not significantly different from resting tension, but changing to pCa 4.5 caused the carotid artery rings to generate 49.5 +/- 4.5% of maximal tension. When a high-tension rigor state was achieved (250 microM ADP, 0 ATP, 0 phosphocreatine, and pCa 9), the addition of 12 mM phosphocreatine effected significant relaxation. These observations implicate an endogenous form of creatine kinase, associated with the myofilaments, which is capable of producing enough ATP for submaximal tension generation and significant relaxation from rigor conditions. These results suggest co-localization of ATPase, MLCK, and creatine kinase on the contractile proteins of the carotid artery. Such an enzymic association may play a role in the energetic supply to the contractile apparatus of vascular smooth muscle.

Adenosine Diphosphate↗

PubMed1983

Transformation of human skeletal muscle cells by simian virus 40.

Molecular studies of the biochemical alterations involved in human myopathies have been restricted because of the finite life-span and slow growth rate of cultures derived from primary tissue. Because the tumor virus simian virus 40 (SV40) can alter both the growth properties and longevity of human cells, we have infected skeletal muscle cultures derived from four biopsies with a small-plaque variant of SV40 and analyzed the biological and biochemical properties of cloned myoblast derivatives. At early times after infection, myoblasts fused normally into multinucleated myotubes, and both unfused and fused cells contained SV40 tumor antigen (T antigen). After six to eight subcultures after infection, the ability of myoblasts to fuse diminished, and clonal cell lines were generated with increased growth rates and saturation densities. Transformed cultures also lost contact inhibition of growth and became anchorage independent. Unlike untransformed myoblasts, SV40-transformed clones did not undergo an increase in creatine kinase activity or a transition of creatine kinase isoenzymes from the BB form to the muscle-specific MM form. Analysis of the pattern of SV40 DNA integration by Southern blotting hybridization analysis in two cloned SV40-transformed myoblast cell lines (KJ-SV40 and PK-SV40) indicated that KJ-SV40 contained at least one site of SV40 DNA integration into chromosomal DNA and PK-SV40 contained at least three sites of SV40 DNA covalently linked to cellular DNA. Cell lysates and growth medium from PK-SV40 transformants contained infectious small-plaque variant SV40, whereas KJ-SV40 did not contain or produce detectable virus. These studies demonstrate that human myoblasts can be immortalized by SV40. This procedure may prove useful for generating large quantities of genetically deficient human cells for biochemical and molecular analysis.

Antigens, Viral, Tumor↗

PubMed1989

Alterations in the creatine kinase system in the myocardium of cardiomyopathic hamsters.

Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.

Animals↗

PubMed1977

Brain isoenzyme of creatine kinase. I. Purification of rabbit enzyme and production of specific antibodies.

Creatine kinase (EC 2.7.3.2) of rabbit brain (BB) was purified to hemogeneity as judged by sedimentation velocity analysis in the ultracentrifuge. Nevertheless, the purified enzyme contained as many as seven extraneous protein species when examined by disc electrophoresis in polyacrylamide gel. The purified preparation also showed antigenic heterogeneity when it reacted in agar gel double immunodiffusion analysis with antibodies produced against it in the rooster. At least some of the extraneous proteins appeared to arise from BB during storage. Subsequently BB was purified to hemogeneity by disc electrophoresis in polyacrylamide gel. It was excised from the gel and injected repeatedly with Freund's complete adjuvant into roosters. A single immunoprecipitin line was noted in agar gel double immunodiffusion analysis when partially pure enzyme reacted with the antibodies raised to the electrophoretically pure protein. These antibodies inhibited the enzymatic activity of BB in both soluble and insoluble immune complexes. They did not, however, form immunoprecipitates with or inhibit rabbit muscle (MM) or rabbit hybrid (MB) creatine kinases. The antibodies were therefore specific for BB and did not react with MM or MB.

Animals↗

PubMed1973

A protein that binds specifically to the M-line of skeletal muscle is identified as the muscle form of creatine kinase.

Published information on the properties of two proteins from chicken muscle, creatine kinase (MM-creatine kinase) and an M-line protein, suggested that they might be identical molecules. Different published procedures were used to purify the two proteins to homogeneity, and the properties of the two preparations were compared. Creatine kinase specific activity increased during purification of M-line protein, reaching a value comparable to that of purified MM-creatine kinase. The two proteins migrated identically in two electrophoretic systems and, after electrophoresis, both could be stained for creatine kinase activity. Double immunodiffusion tests with antibody prepared against MM-creatine kinase established the serological identity of the two protein preparations. Immunofluorescent studies showed that antiserum against MM-creatine kinase was bound in a regular pattern at the centers of the A-band regions of isolated myofibrils. These data show conclusively that the M-line protein and MM-creatine kinase are identical.

Amino Acids↗