Creatine kinase (CK; EC 2.7.3.2) is found in a variety of striated and smooth muscles, and the brain, and is an important enzyme regulator of high-energy phosphate production and utilization within contractile tissues. Serum CK activity is measured routinely as a sensitive indicator of injuries to the skeletal muscle and myocardium. CK has three isozymes (CK-MM, CK-MB and CK-BB) in cytoplasm and two isozymes (non-sarcomeric and sarcomeric) in mitochondria. CK isozymes provide more specific information about injured tissue because of their tissue distribution. CK-MM is useful in skeletal muscle diseases, such as muscle dystrophy, CK-MB in acute myocardial infarction (AMI), and CK-BB in brain damage and malignant tumor of the gastro-intestinal tract. CK-MB is measured either by enzyme activity or mass concentration and is measured as a marker not only in the diagnosis of AMI but also in suspected AMI and unstable angina. Mitochondrial CK, a useful indicator for pinpointing the severeness of muscle injuries, and CK-linked immunoglobulin are recognized as macro-CK because of their large molecular size.

We investigated whether parathyroid hormone-related peptide (PTH-rP), recently found expressed in the heart, exerts growth and contractile effects on adult cardiomyocytes from rat hearts. Synthetic PTH-rP peptides were used covering either a protein kinase C (PKC)-activating domain [PTH-rP(107-111)], or an adenylate cyclase activating domain [PTH-rP(1-34) and PTH-rP(7-34)]. PTH-rP(107-111) (1 micro M) increased creatine kinase BB activity (CK-BB), a CK isoform re-expressed during cardiac hypertrophy, within 24 h by 62+/-12%. This induction was abolished in the presence of the mitogen-activated-protein (MAP)-kinase-kinase inhibitor PD 98059. PTH-rP(107-111) activated p42-MAP-kinase within 15 min, increased protein synthesis (19+/- 4%), total protein mass (19+/-5%), cell volume (45+/-7%), and cross-sectional area (38+/-9%) of cardiomyocytes. Activation of p42-MAP-kinase and increase in protein synthesis were abolished in presence of bisindolylmaleimide, a PKC inhibitor. PTH- rP(107-111) did not directly influence contractile activity but reduced the contractile response to isoprenaline. In contrast, PTH-rP(1-34) and PTH-rP(7-34) induced spontaneous contractile activity in 3-day-old cultures. This induction was abolished in presence of Rp-cAMPS, a protein kinase A inhibitor, indicating an involvement of cAMP in this response. PTH-rP(1-34) also increased the cellular accumulation of cAMP. It is concluded that PTH-rP exert direct effects on adult cardiomyocytes by activating either PKC via a functional domain covered by amino acids 107-111 or by activation of cAMP-dependent protein kinase via a functional domain covered by amino acids 7-34. Since these parts of PTH-rP have either no homology [PTH-rP(107-111)] or only a limited structural similarity [PTH-rP(7-34)] to parathyroid hormone, these activities of PTH-rP have to be clearly distinguished from those described for parathyroid hormone.

To estimate brain damage after cardiac arrest, the concentrations of neuron specific enolase (NSE), GTP-binding protein (G0 alpha), 28 kDa calbindin-D, S100b protein, and creatine kinase BB (CK-BB) in serum and cerebrospinal fluid (CSF) were determined by enzyme immunoassays. Ten mongrel dogs were subjected to 30 min of circulatory arrest at normal body temperature and serial CSF and blood samples were taken during the first 18 h after reperfusion. The NSE concentration in CSF increased significantly after reperfusion, reaching a 15-fold increase (243 +/- 107 ng/ml, p < 0.01) 18 h later, however, it did not increased significantly in serum (8.1 +/- 3.3 ng/ml vs. 23.5 +/- 7.0 ng/ml). G0 alpha concentration in CSF increased sharply between the 2nd and 4th h after reperfusion and peaked 18 h after reperfusion (428 +/- 195 pg/ml, p < 0.01), however, it did not increase significantly in serum. Calbindin-D concentration in CSF increased between the 1st and 6th h after reperfusion, and reached a plateau thereafter (621 +/- 235 ng/ml, a 23-fold increase, p < 0.05) and also increased significantly in serum (p < 0.05). The S100b concentration in CSF also increased dramatically after the 4th h of reperfusion and reached a plateau at the 8th h after reperfusion (16.0 +/- 9.3 ng/ml, a 50-fold increase, p < 0.01), however, it in serum was below the detection threshold. The CK-BB concentration in CSF peaked 4 h after reperfusion (113 +/- 69 ng/ml, a 19-fold increase, p < 0.01) and it in serum increased 4-fold (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac toxicity of preparative regimen (PR) containing high-dose Cyclophosphamide (120 mg/kg) followed by hematopoietic cell transplantation (HCT) was evaluated with 6 biomarkers of cardiac injury: N-terminal pro brain natriuretic peptide (NT-proBNP), creatine kinase MB (CK-MB mass), cardiac troponins (cTnT, cTnI), heart-type fatty acid binding protein (H-FABP), glycogen phosphorylase BB (GPBB). Twenty-three patients (mean age 44.5+/-10.6 years, 15 males) with acute leukemia were studied. All biomarkers were measured the day before PR, the day after PR, the day after HCT and 14 days after HCT. We found NT-proBNP elevations above 500 ng/L in 6 (26.1 %) patients after PR, in 9 (39.1 %) after HCT and in 7 (30.4 %) 14 days after HCT. GPBB became elevated (above 7.30 microg/L) in 5 (21.7 %) patients after PR, remained elevated in 5 (21.7 %) after HCT and in 2 (8.7 %) 14 days after HCT. A significant correlation between elevation in NT-proBNP and GPBB was found. Other markers remained within the reference range early after PR and HCT. Our findings show that administration of PR and HCT for acute leukemia is associated with acute neurohumoral activation of cardiac dysfunction (significant rise in NT-proBNP) and may lead to GPBB elevation. These changes could indicate acute cardiac toxicity due to treatment and require further follow-up. The predictive value for development of cardiomyopathy in the future is unclear. Further studies will be needed to define the potential role of new biomarkers in this context.

OBJECTIVE

To assess cardiac toxicity of anthracycline treatment with six biomarkers of cardiac injury: myoglobin, creatine kinase MB (CK-MB mass), cardiac troponin T (cTnT), cardiac troponin I (cTnI), heart-type fatty acid binding protein (H-FABP), glycogen phosphorylase BB (GPBB).

METHODS

We evaluated anthracycline-induced cardiotoxicity in 12 acute myeloid leukemia patients (mean age 51.3-/+10.7 years, 7 females). All biomarkers were measured at the baseline, after first chemotherapy (CT) with anthracyclines, after last CT with anthracyclines (total cumulative dose 479.8-/+106.2 mg/m2) and 6 months thereafter. Values above the reference range were considered elevated.

RESULTS

GPBB increased above the cut-off (7.30 microg/L) in 2 (16.7%) patients after first CT, in 3 (25.0%) patients after last CT and remained elevated in 2 (16.7%) patients within 6 months after CT. CTnI became elevated (above 0.40 microg/L) in 1 (8.3%) patient after first and last CT and within 6 months after CT. CTnT remained negative (below 0.01 microg/L) during CT in all patients. Six months after CT, delayed cTnT positivity was found in 1 (8.3%) patient. All patients with cTnI or cTnT positivity had elevated GPBB. Other biomarkers (myoglobin, CK-MB mass, H-FABP) remained within the reference range in all patients.

CONCLUSIONS

Our preliminary results suggest that GPBB could be a new promising marker for detection of anthracycline-related cardiotoxicity and probably superior to cardiac troponins. The predictive value for development of cardiomyopathy in the future is not clear and will be evaluated during a prospective follow-up.

OBJECTIVE

Disturbances in venous outflow from the cerebral circulation may result in brain injury. Severe increases in brain venous pressure lead to brain ischemia and, subsequently, brain edema and intracranial hemorrhages. The purpose of this study was to determine the effect of changes in jugular venous bulb pressure (JVBP) on plasma blood brain-barrier biomarkers concentration and disturbances in arteriovenous total and ionized magnesium (a-vtMg and a-viMg) in brain circulation in patients undergoing coronary artery bypass grafting surgery (CABG) with cardiopulmonary bypass (CPB).

METHODS

Prospective observational study.

METHODS

Department of Cardiac Surgery at a Medical University Hospital.

METHODS

Ninety-two adult patients undergoing elective CABG with CPB under general anaesthesia were studied.

METHODS

Central venous pressure (CVP) was measured using a pulmonary artery catheter. The right jugular vein was cannulized retrogradely for jugular venous bulb pressure (JVBP) measurement. Concentrations of plasma S100β protein, matrix metalloproteinase 9 (MMP-9), creatine kinase isoenzyme BB (CK-BB) a-vtMg and a-viMg were measured as the markers of blood-brain barrier dysfunction. All of them were analyzed in comparison with JVBP during surgery and the early postoperative period.

RESULTS

Elevated JVBP was noted after CPB and after surgery. Its increase above 12 mmHg intensified release of S100β, MMP-9 and CK-BB as well as disorders in a-vtMg and a-viMg. CVP correlated with JVBP, S100β, and MMP-9. Moreover, JVBP correlated with S100β and MMP-9.

CONCLUSIONS

Cardiac surgery increased JVBP, and JVBP elevated above 12 mmHg intensified an increase in biomarkers of plasma blood-brain barrier disruption.

BACKGROUND

Central nervous system (CNS) metastases occur in approximately 35% of patients with breast cancer. Parenchymal brain metastases (MET) remain undetected in a large proportion of patients, and only 50% of patients with leptomeningeal carcinomatosis (MC) are diagnosed in vivo.

METHODS

Cytology and activity of the tumor markers tissue polypeptide antigen (TPA) and creatine kinase-BB isoenzyme (CK-BB) were evaluated in the cerebrospinal fluid (CSF) in 71 consecutive patients with breast cancer suspected for CNS metastases.

RESULTS

Forty-three patients had no CNS metastases, 12 patients had MET, 5 patients had both MET and MC, and 11 patients had MC alone. Seven of the patients with MC had an intracerebroventricular (ICV) reservoir inserted, and an additional 70 ICV CSF samples from these patients were obtained. In CSF obtained by lumbar puncture, 11% of the samples were classified as "suspicious for malignancy," but a very limited interobserver variability was demonstrated (Kappa test value, 0.81; 95% confidence limits, 0.67-0.95%). Fifty-one percent of the ICV CSF samples were classified as "suspicious for malignancy" (Kappa test value, 0.58; 95% confidence limits, 0.34-0.82%). TPA and CK-BB were both measured in 101 CSF specimens (61 from lumbar puncture and 40 ICV samples). The differences between patients with and without CNS metastases were significantly different according to TPA (P < 0.00001) and CK-BB (P < 0.00003; Mann-Whitney test). The sensitivity and predictive value of a negative test for having any CNS metastases (in case of elevated values of either TPA or CK-BB or both) were 85% (95% confidence limits, 65-96%) and 90% (95% confidence limits, 76-97%), respectively. In addition, a significant correlation between TPA and CK-BB was demonstrated in CSF from lumbar puncture (Spearmans-Rho, 0.49; P < 0.0001) and ICV (Spearmans-Rho, 0.37; P < 0.02).

CONCLUSIONS

Cytologic evaluation of CSF obtained by lumbar punctures is a reliable procedure. In CSF from ICV reservoirs, cytologic evaluation is of limited use, but CK-BB and TPA is of potential value.

OBJECTIVE

We tested the hypothesis that long-term beta-blocker treatment with bisoprolol prevents creatine kinase (CK) and lactate dehydrogenase system changes that occur after chronic myocardial infarction.

BACKGROUND

The mechanism of the beneficial effect of beta-blocker therapy is still unclear.

METHODS

Six groups of rats were studied. Sham operated (sham) and hearts with ligated left anterior descending coronary artery (myocardial infarction) were untreated, treated early (beginning 30 min after infarction) or treated late (beginning 14 days after infarction). After 8 weeks, hearts were isolated and buffer perfused isovolumetrically. With a left ventricular balloon, mechanical function was recorded at an end-diastolic pressure of 10 mm Hg. Biopsy samples of noninfarcted left ventricular tissue were taken. Enzyme activities were measured spectrophotometrically; isoenzymes were separated by agar gel electrophoresis; and total creatine levels were measured with high performance liquid chromatography.

RESULTS

The decrease in left ventricular developed pressure in untreated hearts (120 +/- 9 vs. 104 +/- 5 mm Hg [mean +/- SE], p < 0.05, sham vs. myocardial infarction) after myocardial infarction was prevented by early treatment (118 +/- 9 vs. 113 +/- 4 mm Hg). Late treatment failed to improve mechanical function. Reduction of CK activity occurring in untreated infarcted hearts (6.4 +/- 0.3 vs. 5.1 +/- 0.3 IU/mg protein, p < 0.05, sham vs. myocardial infarction) was prevented by early beta-blocker therapy. The increase in CK isoenzyme BB and MB levels, decrease in mitochondrial CK isoenzyme levels and increase in anaerobic lactate dehydrogenase isoenzyme levels in untreated infarcted hearts did not occur during bisoprolol treatment. The decrease in total creatine levels after myocardial infarction (74.2 +/- 4.9 vs. 54.9 +/- 3.3 nmol/mg protein, p < 0.05, sham vs. myocardial infarction) was prevented by bisoprolol treatment. Early treatment was more effective than late therapy in preventing CK and lactate dehydrogenase system changes. In addition, in sham hearts, a 40% increase of creatine levels above normal levels was detected.

CONCLUSIONS

Bisoprolol prevented changes in CK and lactate dehydrogenase system that occur after myocardial infarction. These observations may be related to the beneficial effects of long-term beta-blocker treatment in patients with chronic myocardial infarction.

Four human small-cell gastrointestinal carcinoma cell lines were established from tumor tissues of patients with esophageal, gastric or rectal cancer, and were studied morphologically and biochemically in comparison with small-cell lung carcinoma (SCLC) cell lines and common gastric cancer cell lines. Cells from all the small-cell gastrointestinal carcinoma lines were as small as classic SCLC cells and had characteristic neurosecretory granules. Cells from only one line grew as tightly packed spherical aggregates of floating cells, and those of the other 3 grew attached to substrate. Although high levels of creatine kinase brain isoenzyme (CK-BB) were detected in all 4 cell lines, 2 of them showed low levels of aromatic L-amino-acid decarboxylase and 3 had low levels of neuron-specific enolase (NSE). None of the lines showed simultaneous elevation of enzymes. C-myc, N-myc, and L-myc were not amplified in any of the cell lines, but c-myc mRNA was expressed in 2 lines. Our findings indicate that all small-cell gastrointestinal carcinoma cells examined belong to the variant type which is used in the classification of SCLC. Furthermore, the ECC18 line, derived from esophageal cancer, seemed to be of true endocrine cell origin, while the 3 other small-cell gastrointestinal carcinoma lines seemed to arise via neoplastic neometaplasia from adenocarcinoma cells to endocrine cells.

For the determination of their possible utility as tumors markers, 2 neural-associated isozymes, neuron-specific enolase [(NSE) EC 4.2.1.11] and creatine kinase BB [(CK-BB) EC 2.7.3.2], were quantitated by radioimmunoassay in human neuroectodermal-derived cell lines, primary brain tumors, and sera and cerebrospinal fluid (CSF) from brain tumor patients. The NSE content of neuroblastoma cell lines was more than sixfold that of the glioma and medulloblastoma lines; the CK-BB content of neuroblastoma and medulloblastoma lines was fourfold to nineteen-fold that of the glioma and other lines. Expression of NSE in neuroblastoma cell lines was not related to time in culture and was cell line specific. NSE in ex vivo medulloblastomas was raised six to ten times that in astrocytomas and gliomas, although no significant differences were noted for the CK-BB content. Serum and CSF NSE levels were markedly raised above control values in 10 of 29 and 6 of 10 cases of astrocytoma, respectively. Raised CK-BB levels in serum (greater than 10 ng/ml) and CSF (greater than 12 ng/ml) were found in 9 of 18 and 2 of 10 patients, respectively. These data suggest that NSE is preferentially expressed by neuroblastoma lines and medulloblastomas and that NSE and CK-BB may have clinical utility as markers for prognosis, diagnosis, and monitoring of response to therapy.

In skeletal muscle isoenzymes of CK were determined by immunprecipitation and chromatography. The activity of CK-MB was between 17 and 47 U/g muscle, corresponding to a quota between 2,1 and 4,2% of the total activity. In sera of patients with muscular dystrophy, polymyositis, hypothyroidism, after arterial embolism, epilepsy, hyperventilation, operations and polytrauma with and without injury to the thorax isoenzymes were measure by immune precipitation- and immune inhibition-test. The percentage of CK-MB in all sera was less than 6% of the total CK-activity (range: 0 to 6%). Only patients in the first day after neurosurgical operations showed a quota till to 6.5% CK-MB. In serum of patients after polytrauma without injury to the thorax the percentage of CK-MB ranged from 0-5.7% while after polytrauma with injury to the thorax and a reasonable suspicion of a damage to the myocardium this quota was between 5.1 and 23.6% of the total activity. CK-BB activity was not detectable in any cases. Therefore a disease or damage of the skeletal muscle is more probable, if the percentage of CK-MB in less than 6%, because in sera of patients with myocardial infarction in the first 48 h after beginning of the symptoms this quota of CK-MB in the most cases in more than 6%.

We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.

We investigated the redistribution of myocardial isoenzymes of creatine kinase (CK) and lactate dehydrogenase (LD) in rats with right heart failure induced by monocrotaline and assessed the effect of enalapril, an angiotensin converting enzyme inhibitor. Wistar rats were divided into four groups: (1) control (n = 20), (2) control + enalapril (25 mg/kg/day) (n = 22), (3) monocrotaline (50 mg/kg) (n = 45), (4) monocrotaline (50 mg/kg) + enalapril (25 mg/kg/day) (n = 32). After 4 weeks, the monocrotaline group developed severe pulmonary hypertension and right ventricular hypertrophy with marked decrease in myocardial norepinephrine and increase in both plasma atrial natriuretic peptide and mortality rate (33.3%). The marked decrease in both MM and mitochondrial CK ('creatine shuttle') and the relatively constant BB and MB CK caused the net depression of total CK. The depression of LD1 (aerobic LD) was remarkable compared with the relatively constant total LD. In the monocrotaline+enalapril group, mortality rate (9.4%), cardiac hypertrophy and plasma atrial natriuretic peptide were all significantly reduced and myocardial norepinephrine recovered although pulmonary hypertension was not improved at all. However, myocardial total, MM and mitochondrial CK and LD1 activities were all recovered completely or partially in this group. Thus, enalapril reduced cardiac hypertrophy and failure and improved the prognosis in this model of pulmonary hypertension. This beneficial effect of enalapril was not associated with pulmonary vasodepression but with the inhibition of myocardial isoenzyme redistribution of CK and LD, i.e. the preservation of 'creatine shuttle' and aerobic LD.

Serum creatine kinase BB-isoenzyme (CK-BB) activity was studied on the first day of life in 31 acutely asphyxiated infants, 70 infants born after high risk pregnancies (pre-eclampsia or intrauterine growth retardation, or both), and 47 very low birthweight infants. Neuro-developmental evaluation was carried out at 2.2-2.5 years. Eight infants died with, and eight without, hypoxic-ischaemic lesions of the brain, 14 had cerebral palsy, 16 had mild motor impairment, six had developmental delay without motor impairment, and 96 were normal at follow up. Infants who died with brain injury had significantly higher CK-BB activity than infants with normal outcomes (geometric mean 12 U/l); the mean difference was 82 U/l with a 95% confidence interval from 31 to 219 U/l. CK-BB in infants with cerebral palsy and mild motor impairment (geometric means 12 and 15 U/l, respectively) were similar to controls. CK-BB activity after birth is predictive of neonatal death but not of neurological damage in survivors.

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.

We report the expression of the human muscle (CK-MM) and brain (CK-BB) creatine kinases in Escherichia coli. The proteins have been purified to apparent homogeneity and several of their physical and kinetic properties investigated. In the process, we have conclusively verified the correct DNA sequence of the genes encoding the respective isozymes, and determined the correct primary structure and mass of the gene products. Alignment of the primary sequences of these two enzymes shows 81% sequence identity with each other, and no obvious gross structural differences. However, Western blot analyses demonstrated the general lack of antigenic cross-reactivity between these isozymes. Preliminary kinetic analyses show the K(m) and k(cat) values for the creatine and MgATP substrates are similar to values reported for other isozymes from various tissues and organisms. The human muscle and brain CKs do not, however, exhibit the synergism of substrate binding that is observed, for example, in rabbit muscle creatine kinase.

Creatine kinase (CK) is a highly conserved enzyme abundant in skeletal muscle that has a key role in high energy phosphate metabolism. The localization of the muscle isoenzyme of CK (MM-CK) to the M line and the sarcoplasmic reticulum of myofibrils has been suggested to be important for proper force development in skeletal muscle. The importance of this subcellular compartmentation has not been directly tested in vivo. To test the role of myofibrilar localization of CK, the consequences of a complete CK isoform switch from MM-CK to the brain (BB-CK) isoform, which does not localize to the M line, was studied in transgenic mouse skeletal muscle. In MM-CK knockout mice there are large contractile defects. When MM-CK was replaced by BB-CK, the aberrant contractile phenotypes seen in MM-CK knockout mice were returned to normal despite the lack of myofibrillar localization. These results indicate that CK compartmentation to the myofibril of skeletal muscle is not essential for contractile function and that there is functional equivalence of creatine kinase isoforms in supporting cellular energy metabolism.

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 chromatofocusing 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-terminal 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.

In this two-step automated assay of the MB isoenzyme of creatine kinase (CK-MB), developed for the Abbott "IMx" immunoassay analyzer, monoclonal anti-CK-MB antibody immobilized onto latex microparticles and polyclonal anti-CK-MM antibody coupled to alkaline phosphatase are used. Within-run CVs ranged from 3.9% to 9.0%, between-run CVs from 0.0% to 5.6%, and the sensitivity was 0.2 microgram/L. Twenty-four results can be obtained in about 37 min. Analytical recovery of CK-MB added to human serum or plasma ranged from 89% to 109%. Icteric, lipemic, or hemolyzed samples did not interfere with CK-MB recovery. Cross-reactivity with CK-MM and CK-BB was 0.012% and 0.001%, respectively. The normal reference interval was 0-5 micrograms/L. IMx CK-MB results correlated well with CK-MB enzyme activity as determined by electrophoresis (n = 203; r = 0.97; slope = 0.90; y-intercept = -4.29) and with commercial immunoassays. We think that this assay will be useful for confirmation of acute myocardial infarction, both in critical-care units and in the clinical laboratory.

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.

Several enzymes that occur in multimolecular forms undergo transitions during myogenesis. Studies of such developmentally regulated isozymes (e.g. creatine kinase) indicate that muscle cells, cultured in the absence of neural tissue never develop fully mature isozyme patterns, but continue to express large amounts of 'housekeeping' isozymes that are characteristically present in fetal muscle. We studied two developmentally controlled isozymes, creatine kinase (CK) and phosphoglycerate mutase (PGAM) in normal human muscle, both aneurally cultured and co-cultured with fetal mouse spinal cord complex. Innervated cultures attain a greater degree of maturity than non-innervated cultures, as revealed by light and electron microscopy, showing well-developed sarcomeres and motor endplates after several weeks in vitro. During early stages of muscle regeneration in co-culture, characteristic fetal isozyme patterns of CK-BB and PGAM-BB activity predominate, as in aneural cultures. The muscle-specific isozymes (CK-MM; PGAM-MM) begin to appear as the muscle differentiates, and after 2-3 months in co-culture only, virtually all enzyme activity is due to the muscle-specific forms of CK and PGAM, as is normally observed in mature skeletal muscle in vivo.

For one to obtain a precise estimate of creatine kinase (CK) activity in cerebrospinal fluid, the sample fraction is increased by about 10-fold over that used for serum. This increases the concentration of interfering substances, Ca being especially important. Therefore, the relationship between Ca, Mg, and EDTA was examined. Enzyme activity was maximal with 15 mmol of Mg per liter in the presence of 3 mmol of EDTA per liter, otherwise according to the (Scandinavian) recommended conditions for determination of CK activity in serum. These modifications increased the activity of CK by 35% for CK-MM and by 60% for CK-BB. Counteraction of Ca-induced inhibition was the main reason to this increase. We describe a practical and sensitive method for determining CK in cerebrospinal fluid.

A full-length human creatine kinase B (B-CK) cDNA was used to produce a recombinant baculovirus (AcDZ1-BCK). Sf9 cells infected with this recombinant expressed a homodimeric protein composed of 43 kDa subunits which, under optimal conditions, formed up to 30% of the total soluble cellular protein. Upon analysis by PAGE, zymogram assay and gel filtration chromatography the recombinant protein behaved like authentic dimeric human BB-CK protein. Studies with a newly produced monoclonal antibody (CK-BYK/21E10) directed against an epitope in the N-terminus of the protein confirmed the identity of the product. The recombinant BB-CK protein was purified to over 99% homogeneity from the total protein extract of AcDZ1-CKB infected cells in one single step involving anion exchange column chromatography on MonoQ in FPLC. Dialysed protein had a specific activity of 239 U/mg protein.