Creatine Kinase Subunits Research Articles

Immunohistochemical study on the distribution of creatine kinase subunits in early human embryos.

Creatine kinase (CK) is a dimer composed of two immunologically distinct subunits, CK-M and CK-B, and takes part in the production of adenosine triphosphate. In the present study, the distribution of CK in the muscles and the nervous system was almost identical to that reported in older fetuses. However, in the epithelia of trachea and esophagus, CK-B immunoreactivity was apparent only in young embryos and decreased with the age of the embryo, while CK-M immunoreactivity was absent or weak in early stages and increased thereafter. In the surface ectoderm, CK-B was not detected immunohistochemically while CK-M was evident in early embryos and decreased as the embryonic stage advanced. Among the ectodermal derivatives, the lens placode was immunoreactive only to CK-M antibody at stage 13. This immunoreactivity decreased and finally disappeared along with the formation of the lens vesicle. In the urogenital system, mesonephric tubules were immunoreactive to both CK-B and CK-M antibodies, while mesonephric ducts were immunoreactive only to the CK-M antibody. Mesonephric glomeruli were not immunostained with these antibodies. These results in the urogenital system were found in all embryos examined. The present results suggest that the distribution of CK subunits in several organs of earlier embryos differs not only from that in adults, but also from that in older embryos and fetuses.

Zonal and Age-Related Difference in the Amounts of Creatine Kinase Subunits in Cartilage

Energy metabolism in cartilage may affect the morphogenetic events of skeletal growth. Investigating enzymes responsible for energy metabolism in cartilage, such as creatine kinase (CK), can provide clues to an understanding of pathogenesis and treatment of osteochondrodysplasias. In this study levels of CK subunits M (muscle type) and B (brain type) were measured by a highly sensitive enzyme immunoassay system in growth and resting cartilages of the rat rib at various ages. CK-M was predominant, but there was no statistically significant difference in its quantity of CK among cartilages of various ages or between resting and growth cartilage. In contrast, although CK-B levels were low, they showed a significant decrease with advancing age and a significant increase in growth cartilage as compared with resting cartilage. The results of this study suggest that CK in cartilage, especially CK-B, may play an important role in skeletal growth.

Dynamics of creatine kinase shuttle enzymes in the human heart

Myocardial cytoplasmic creatine kinase subunits M and B, mitochondrial CK (CKMIT), and citrate synthase (CS) were determined in 10 locations of the normal human heart (n = 8) and in papillary muscles of patients operated on for mitral regurgitation (n = 6). Compared to atrial biopsies, septal and left ventricular biopsies showed higher activities for CS (P less than 0.0001), total CK (P less than 0.05) and CKMIT (P less than 0.0001). CKM was evenly distributed. CKB activity in the right septum and left ventricular locations were 0.5-1% of total CK and 4-5 times lower than those of the atria and the right ventricular free wall. Activities of CS, CKB and CKMIT in right septal biopsies did not differ from those in left ventricular locations. The activities of CS, total CK, and CKM in papillary muscle from patients operated on for mitral regurgitation did not differ from that of healthy papillary muscle. CKMIT was about 40% lower (P less than 0.02), whereas CKB was 15-20 times higher (P less than 0.0001) than in the healthy heart. In conclusion, adaptations within the creatine kinase system occur in the human heart in health and disease. Small amounts of CKB in the normal left ventricle, as opposed to the right ventricular free wall, might be related to differences in myocardial perfusion during the cardiac cycle. In disease, a decreased CKMIT and dramatically increased CKB may indicate a stressed intracellular energy transfer. CK enzyme activities in right septal biopsy specimens may be used as an indication of metabolic stress on the myocardium of the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of expression of M, B, and mitochondrial creatine kinase mRNAs in the left ventricle after pressure overload in rats.

Pressure overload of the left ventricle induces synthesis of creatine kinase isoenzymes. To determine whether this response is associated with an altered pattern of creatine kinase gene expression, we induced arterial hypertension in rats by suprarenal aortic banding. After 4 days, left ventricular myocardium from hypertensive (n = 7) and normotensive, sham-operated (n = 5) rats was analyzed for isoenzyme activities by chromatography; M and B creatine kinase subunit protein by Western blot; and M, B, and mitochondrial creatine kinase mRNA by Northern blot. Although total creatine kinase activity increased in hypertensive (1,096 +/- 214 IU/g left ventricle) compared with normotensive rats (648 +/- 81 IU/g left ventricle, p less than 0.01), the relative proportions of the cytoplasmic and mitochondrial isoenzymes did not change. The mass of M and B subunits increased 1.9- and 2.7-fold, respectively, in hypertensive compared with control rats. Similarly, the mRNA for M and B subunits as well as mitochondrial creatine kinase increased 2.6-, 1.6-, and 1.8-fold, respectively, in hypertensive rats compared with control rats. Thus, increased energy requirements in acute pressure overload are met by generalized induction of creatine kinase mRNA and subunit protein and not by an isoenzyme switch.

Physical and genetic mapping of a novel chromosome 19 ERCC1 marker showing close linkage with myotonic dystrophy

Recent genetic linkage analyses have mapped the myotonic dystrophy locus to the region of 19q13.2–13.3 lying distal to the gene for creatine kinase subunit M (CKM). The human excision repair gene ERCC1 has also been mapped to this region of chromosome 19. A novel polymorphic DNA marker, pEO.8, has been isolated from a chromosome 19 ERCC1-containing cosmid that maps to a 300-kb NotI fragment encompassing both CKM and ERCC1. Genetic linkage analysis reveals close linkage between pEO.8 and myotonic dystrophy (DM) ( z max = 19.3, θ max = 0.01). Analysis of two key recombinant events suggests a mapping of DM distal to pEO.8 and CKM.

Immunohistochemical Study of Creatine Kinase Isozymes in the Human Upper Limb Bud

Creatine kinase (CK) is involved in the production of ATP and is composed of two immunologically distinct subunits, B (CK-B) and M (CK-M). In the differentiation of myoblasts, the isozyme of CK changes from CK-B to CK-M. In the present study, the expression of CK subunits was studied immunohistochemically in the upper limb bud of human embryos (Carnegie stages 13-21). It was found that CK-B and CK-M immunoreactive cells appeared at stage 15 and at stage 18, respectively.

A novel creatine kinase cDNA whose transcript shows enhanced testicular expression

A cDNA (TCK1) encoding a creatine kinase (CK) subunit has been isolated from a cDNA library prepared from rainbow trout testis poly(A) + RNA. The predicted amino acid sequence for this CK subunit showed a novel amino-terminal coding region. Northern blot analyses demonstrated preferential expression of this CK subunit in testis relative to other CK containing organs. Southern blot analysis suggested a single copy number for this CK gene.

Phosphorylation of chicken brain-type creatine kinase affects a physiologically important kinetic parameter and gives rise to protein microheterogeneity in vivo

In addition to the two monomer subunits of chicken brain-type creatine kinase (B-CK, EC, 2.7.3.2), termed Bb (basic) and Ba (acidic), another subspecies called Bb ∗ was identified by chromatofocussing in the presence of 8 M urea (Quest et al., [20]). The latter low abundance protein species, isolated from tissue extracts, comigrated on 2D-gels with three minor species (Bbl-3), initially identified in immunoprecipitated, [ 35S]methionine labeled in vitro translation products of cDNA coding for the basic monomer Bb. During in vitro translation experiments in the presence of [ 32P]-γ -ATP, Bbl-3 were labeled while phosphatase treatment eliminated these minor species. It is concluded that Bb * is identical to Bbl-3 and represents phosphorylated derivatives of Bb. B-CK dimer populations from different tissues were separated by ion-exchange chromatography and the K m values of the resulting fractions were determined under phospho-creatine (CP)-limiting conditions. In fractions containing only Bb and Bb ∗ two kinetically different enzyme species were detected ( K m values for CP = 1.6 mM and 0.8 mM), while fractions containing B-CK dimers composed of the major Ba and Bb monomers, but no Bb ∗, were homogeneous in this respect ( K m for CP = 1.6 mM). Phosphorylation of Bb to yield Bb ∗ is concluded to reduce the K m of B-CK dimers for CP by about 50%. This K m shift is within the range of CP concentrations found in tissues expressing the B-CK isoform and may therefore be of physiological relevance.

Is the subunit the minimal function unit of creatine kinase?

The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M′M′) and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M′B),as isolated by PAGE, has the same K m for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB′) has half the activity of the native muscle enzyme. The M′B, MB′ and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M″B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide and the native brain enzyme subunit. The above result suggest strongly the independent catalytic function of the subunit of creatine kinase.

Separate nuclear genes encode sarcomere-specific and ubiquitous human mitochondrial creatine kinase isoenzymes.

Creatine kinase (EC 2.7.3.2) isoenzymes play a central role in energy transduction. Nuclear genes encode creatine kinase subunits from muscle, brain, and mitochondria (MtCK). We have recently isolated a cDNA clone encoding MtCK from a human placental library which is expressed in many human tissues (Haas, R. C., Korenfeld, C., Zhang, Z., Perryman, B., Roman, D., and Strauss, A. W. (1989) J. Biol. Chem. 264, 2890-2897). With nontranslated and coding region probes, we demonstrated by RNA blot analysis that the MtCK mRNA in sarcomeric muscle is distinct from this placenta-derived, ubiquitous MtCK cDNA. To compare these different mRNAs, a MtCK cDNA clone was isolated from a human heart library and characterized by complete nucleotide sequence analysis. The chemically determined NH2-terminal 26 residues of purified human heart MtCK protein are identical to those predicted from this sarcomeric MtCK cDNA. The human sarcomeric and ubiquitous cDNAs share 73% nucleotide and 80% predicted amino acid sequence identities, but have less than 66% identity with the cytosolic creatine kinases. The sarcomeric MtCK cDNA encodes a 419-amino acid protein which contains a 39-residue transit peptide essential for mitochondrial import. Primer extension analysis predicts a 348-base pair 5'-nontranslated region. RNA blot analysis demonstrates that heart-derived MtCK is sarcomere-specific, but the ubiquitous MtCK mRNA is expressed in most tissues. Thus, separate nuclear genes encode two closely related, tissue-specific isoenzymes of MtCK. Our finding that multiple genes encode different mitochondrial protein isoenzymes is rare.

Two different B-type creatine kinase subunits dimerize in a tissue-specific manner

Brain-type creatine kinase B-CK (EC 2.7.3.2) was purified from several chicken tissues, e.g. cardiac muscle, brain, gizzard and retina. Two major monomeric chicken B-CK subunits, designated Bb (basic) and Ba (acidic), which differ in isoelectric point, were separated by chromatofocussing in the presence of 8 M urea on a MonoP column. The two subunits were shown by peptide mapping, amino acid analysis and partial sequencing, as well as by immunological criteria, to be distinct B-CK polypeptides. The N-tenninal sequence of 30 amino acid residues of Bb correspond entirely to data derived from a B-CK c-DNA clone termed H4 [(1986) Nucleic Acids Res. 14, 1449-1463], whereas the N-terminus of the acidic Ba species was blocked. Native dimeric B-CK isoenzymes obtained from these tissues were separated by ion exchange chromatography on a MonoQ column yielding two B-CK dimer populations, type-I and type-II B-CK, varying in relative proportions. Quantitation of the CK activity peak ratios of these two populations revealed the existence of a tissue-specific, post-translational mechanism regulating B-CK dimerization in neural tissues. Tissue-specific dimerization of the two distinct B-CK monomer species may represent a means of specifying the intracellular distribution of the dimeric B-CK subspecies.

Resonance energy transfer between the active sites of rabbit muscle creatine kinase: analysis by steady-state and time-resolved fluorescence.

Resonance energy transfer between the reactive thiols of rabbit muscle creatine kinase was evaluated. The reactive thiols are located at the active site, one occurring on each subunit of the dimeric protein that is known to be a constituent of the M-line structure of the myofibril. Transfer efficiency was evaluated from energy donor quenching of fluorescence by steady-state and phase-modulation lifetime measurements and determination of sensitized emission of the acceptor. Several sulfhydryl-specific donor fluorophores were used including 5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, 7-(dimethylamino)-3-(4-maleimidylphenyl)-4-methylcoumarin, and 2-[4-(iodoacetamido)anilino]naphthalene-6-sulfonic acid (IAANS). Energy transfer acceptors included 5-(iodoacetamido)fluorescein and the nonfluorescent dye [4-[[4-(dimethylamino)phenyl]azo]phenyl]iodoacetamide. In order to prepare the necessary homodimer labeled with both donor and acceptor, advantage was taken of the biphasic reaction between creatine kinase and IAANS. In some instances, donor/acceptor hybrids were prepared by denaturation/renaturation procedures, and possible deviations from expected hybridization stoichiometry were considered. Disproportionation of singly labeled dimers (to unlabeled and doubly labeled dimers) was not observed when the brain isozyme of creatine kinase was used to trap dissociated dye-conjugated or unlabeled muscle-type subunits of creatine kinase. From studies of five different donor/acceptor combinations, the efficiency of energy transfer was found to occur over a range of 5-14%, indicating that the reactive thiols are well separated. Overlap integrals and quantum yields were evaluated, and estimates of the range of orientation factor were obtained to determine a range for the distance between the active sites of creatine kinase. When the ranges are overlapped, a limited distance of 48.6-60.4 A is obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of MB creatine kinase isoform conversion in vitro and in vivo in dogs.

Time-dependent removal of the COOH-terminal lysine residue from each subunit of tissue MM creatine kinase by plasma carboxypeptidase N produces two additional isoforms that are readily separated, thereby permitting sensitive, early detection of acute myocardial infarction. Only two isoforms of MB creatine kinase have been detected in plasma leading to speculation that the COOH-terminal lysine on the B subunit is resistant to hydrolysis. To define the biochemical changes resulting in MB creatine kinase isoform conversion, we incubated highly purified MB creatine kinase from canine myocardium with plasma carboxypeptidase N. Quantitative anion-exchange chromatography of incubation mixtures and serial plasma samples from dogs subjected to coronary occlusion revealed a second, more acidic form evolved with time that was separated from the tissue isoform. Cyanogen bromide digestion of the two isoforms followed by amino acid sequencing of COOH-terminal peptides showed that MB creatine kinase undergoes removal of the COOH-terminal lysine residue from both M and B subunits. An intermediate form lacking lysine on the M subunit was delineated during incubations by the combined use of anion-exchange chromatography and conventional electrophoretic techniques. Thus, sequential cleavage of lysine from subunits of MB creatine kinase produces an intermediate isoform that has not been detected previously because of difficulties separating it from the tissue and fully converted isoforms.

Five-year mortality rate in relation to enzyme-estimated infarct size in acute myocardial infarction

In 727 patients with acute myocardial infarction, different enzyme variables reflecting infarct size were related to the 5-year mortality rate. The maximum activity of serum heat-stable lactate dehydrogenase (LD), analyzed every 12 hours for 48 to 108 hours, was significantly associated with the 5-year mortality rate when patients with a first myocardial infarction were evaluated ( p < 0.001), and similarly ( p < 0.001) when patients with a previous myocardial infarction were included in the analyses. Very similar results were found when the maximum activity of aspartate aminotransferase (ASAT) analyzed once daily for 3 days was related to the mortality rate over 5 years, whereas the maximum activity of creatine kinase (CK) and CK subunit B analyzed every 6 hours for 48 hours in a subset of patients did not predict the outcome to the came extent. The results from LD and ASAT analyses clearly indicated that the association between infarct size and 5-year mortality rate was caused by the much higher mortality rate in patients with larger infarcts during the first year after onset of infarction, whereas after the first year, incidence of death appeared to be independent of the original infarct size. Thus we conclude that although a highly significant relationship between infarct size and overall 5-year survival was found, the mortality rate seemed to be higher in patients with larger infarcts, particularly during the first year after infarction.

Pleomorphic rhabdomyosarcoma in adults: Immunohistochemistry as a tool for its diagnosis

Pleomorphic rhabdomyosarcoma in adults over 30 years of age was a diagnosis frequently made in the 1960s and 1970s. Since the general acceptance of malignant fibrous histiocytoma (MFH) as a tumor entity at the end of the 1970s, however, it has become a very rare tumor in adults. Therefore, 21 cases originally diagnosed on the basis of histology and clinical data as pleomorphic rhabdomyosarcoma in the 1960s and 1970s were reexamined immunohistochemically. Other types of pleomorphic sarcomas involved in the differential diagnosis were also studied. Specific antibodies against vimentin, desmin, creatine kinase subunit M, skeletal muscle actin and myosin, and myoglobin, and the avidin-biotin-peroxidase complex technique were used. The immunohistochemical findings indicate that rhabdomyosarcoma occurs only rarely in adults over 30 years of age and that the majority of the tumors have to be reclassified as MFH or leiomyosarcoma. On the other hand, several pleomorphic sarcomas were found to be diagnosed incorrectly as MFH or liposarcoma by routine histologic stains and electron microscopy. The revised diagnosis was pleomorphic rhabdomyosarcoma for one case and pleomorphic leiomyosarcoma for the other cases. Thus, this study clearly shows the usefulness of immunohistochemistry as a technique in the diagnosis of pleomorphic sarcomas in adults.

Kinetic immunochemical method for the simultaneous quantification of creatine kinase isoenzymes.

We describe the development and evaluation of a kinetic immunochemical method for the simultaneous quantification of isoenzymes. Specifically, we use the inhibition of the M subunit of creatine kinase (CK; EC 2.7.3.2) by antibodies to quantify isoenzymes CK-MM and CK-MB in serum matrices. Nonlinear least-squares data-processing is used to compute the enzyme activities from the time-dependent response of absorbance vs time. Variables affecting the method, namely temperature, substrate concentration, and antibody concentration as well as their interactions, are evaluated by using response-surface methodology. For several concentrations of CK-MM and CK-MB in the range of diagnostic significance, least-squares fits of computed (y) vs expected (x) values yielded equations of y = 0.98x + (2.0 X 10(-5)) s-1 for CK-MM and y = 1.04x - (4.6 X 10(-5)) s-1 for CK-MB for rates between 0 and 3.5 X 10(-3) s-1 (CK-MM) and 0.5 X 10(-3) s-1 (CK-MB). The equations for comparison of kinetic results (y) with results by a kit method (x) were y = 0.97x + (6.5 X 10(-5)) s-1 for CK-MM and y = 1.02x + (4.3 X 10(-5)) s-1 for CK-MB. Pooled day-to-day relative standard deviations (CVs) for the kinetic method were 4.1% and 2.8% for CK-MM and CK-MB, respectively.

Serum creatine kinase and lactate dehydrogenase during cardiac irradiation.

Thirty patients irradiated to a major part of the heart had serial determinations of serum total creatine kinase (S-CK), creatine kinase subunit B (S-CK-B), total lactate dehydrogenase (S-LD) and lactate dehydrogenase isoenzyme 1 (S-LD-1) activity at the beginning, at the end and 1-4 months after radiotherapy. One patient had an elevated total S-CK activity three months after radiotherapy, but none of the patients had an elevated S-CK-B activity during follow-up. Three patients had elevated serum LD before irradiation, two patients during and two patients after radiotherapy, but only one patient had an elevated S-LD-1 activity, which decreased during irradiation. We conclude that the heart muscle is not injured by cardiac irradiation to such an extent that CK, CK-B, LD and LD-1 activities rise in serum, if moderate doses (39-62 Gy, NSD 1,137-1,775 rets) are delivered to the heart.

Highly sensitive enzyme immunoassay for human creatine kinase MM and MB isozymes

A sensitive enzyme immunoassay system for measurement of MM and MB forms of human creatine kinase (CK) was developed using purified monospecific antibodies to the M subunit and to the B subunit of CK. The CK-MM assay system consisted of polystyrene balls with immobilized F(ab') 2 fragments of anti-M and the same antibody Fab' fragments labeled with β- d-galactosidase from Escherichia coli. The CK-MB was assayed with the polystyrene balls with either antibody (anti-M or anti-B) F(ab') 2 fragments and the other antibody (anti-B and anti-M, respectively) Fab' fragments labeled with galactosidase. The assays were highly sensitive and 3 pg of CK-MM and CK-MB were measurable. The CK-MM assay was specific to the M subunit of creatine kinase, and it cross-reacted about 15% with CK-MB, but not with CK-BB. The CK-MB assay did not cross-react with CK-MM nor CK-BB. Therefore, concentrations of CK-MM could be estimated by subtracting the cross-reacting values of CK-MB. Coefficients of variation in within-run and between-run precision studies for serum CK-MM and CK-MB were < 9%. Serum levels in healthy adults of various ages (16–59 yr old) were ranged from 35.2–132 ng/ml for CK-MM and from 0.40–1.77 ng/ml for CK-MB. There was apparently no statistical significance among the sex- and age-related differences. Concentrations of CK-MM and CK-MB in various human tissues were also determined. The CK-MM was present abundantly in the heart and the tissues composed of striated muscles (skeletal muscle, diaphragm and proximal esophagus). The CK-MB was distributed not only in the heart but also in the striated muscle tissues at a relatively high level.

Highly Sensitive Immunoassay for Rat Brain‐Type Creatine Kinase: Determination in Isolated Purkinje Cells

Ultrasensitive enzyme immunoassay method for the measurement of rat brain-type creatine kinase BB (CK-BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK-BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK-B), showing about 10% cross-reactivity with CK-MB, but it did not cross-react with CK-MM and neuron-specific gamma gamma enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK-BB, being sufficiently sensitive for the measurement of CK-B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK-B in a single Purkinje cell was 1.64 pg. The CK-B concentration in rat cerebellum (about 22 micrograms/mg protein) was about twofold higher than that (about 13 micrograms/mg protein) in the cerebrum. High levels (greater than 5 micrograms/mg protein) of CK-B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK-B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.

Application of two dimensional electrophoresis to characterise hormonally sensitive proteins in the normal and abnormal uterus

AbstractProgesterone therapy of androgenized rats results in the elimination of histological abnormalities of the uterus associated with constant unopposed oestrogenic stimulation. Subcellular fractions prepared from uteri of normal, androgenized and progesterone treated animals have been analysed by two‐dimensional gel electrophoresis. In all fractions, protein changes were observed as a result of androgenization and subsequent progesterone therapy. The identity of some of these proteins has been assigned following peptide mapping. Following progesterone therapy, the relative proportion of cytosolic tubulins, tropomyosins and D‐glucose‐6‐phosphate dehydrogenase were increased. In the abnormal uterus, a protein with similarity to the B subunit of creatine kinase was present in the mitochondrial fraction but could not be detected in the cytosol. Subsequent to progesterone therapy, this protein was no longer detectable in the mitochondrial fraction whilst two molecular forms of the B subunit of creatine kinase appeared in the cytosol. These hormonally‐sensitive changes in both structural and enzymatic proteins, which are ubiquitous to most cell types, are discussed in relation to morphological differentiation of the uterus and the molecular mechanisms underlying cell growth.