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PubMed1991

Identification of creatine kinase isoenzymes in the guinea-pig. Presence of mitochondrial creatine kinase in smooth muscle.

Isoenzymes of creatine kinase (CK, EC 2.7.3.2) in guinea-pig smooth, cardiac and skeletal muscles as well as in brain were analyzed by cellulose acetate electrophoresis and FPLC gel permeation chromatography. In crude tissue extracts of smooth muscles brain type BB-CK and the hybrid form MB-CK were detected, but in enriched mitochondrial fractions from different guinea-pig smooth muscles, mitochondrial type Mi-CK was unambiguously identified. Smooth muscle Mi-CK displayed the same electrophoretic mobility as Mi-CK from brain, which migrates slower than cardiac Mi-CK. Identical to parallel experiments with Mi-CK from cardiac muscle and brain, smooth muscle Mi-CK could be resolved into dimeric and octameric species, the latter being remarkably stable. In contrast to guinea-pig smooth muscles, Mi-CK was not detected in chicken gizzard tissue extracts nor in enriched mitochondrial fractions thereof. The presence of Mi-CK, predominantly in octameric form, in guinea-pig smooth muscles, but not in chicken gizzard, may represent a clue for the different physiological properties of these muscles and may provide the molecular basis for the dependence of the PCr production on oxidative metabolism observed in the guinea-pig taenia caeci.

Animals↗
PubMed1994

Hybridization of matrix-bound MM-creatine kinase with BB-creatine kinase and arginine kinase.

Dimeric rabbit muscle creatine kinase (MM-CK) was bound to CNBr-activated Sepharose 4B by one of its subunits (MM-CKA). Treatment of MM-CKA with guanidine hydrochloride released the unbound subunit to yield the matrix-bound monomer (M-CKB). M-CKB recombined with dissociated MM-CK soluble subunits to reconstitute a matrix-bound dimer (MM-CKC). M-CKB also associated with dissociated subunits of BB-CK from crude extracts of rabbit brain and of arginine kinase from sea cucumber muscle (MM-AK) to form the matrix-bound heterohybrids MB-CKC and M-CK/M-AKC, respectively. Guanidine hydrochloride gradient elution studies showed that MM-CKA, MM-CKC and MB-CKC were all dissociated at the same concentration of the denaturant (0.96 M), while the M-CK/M-AKC heterohybrid was less stable, dissociating at 0.5 M. The specific interaction between subunits of echinoderm and mammalian phosphagen kinases to form a hybrid enzyme of dual substrate specificity supports the view that these enzymes had a common evolutionary origin.

Animals↗
PubMed1985

Expression of a rat brain creatine kinase-beta-galactosidase fusion protein in Escherichia coli and derivation of the complete amino acid sequence of rat brain creatine kinase.

Expression of a rat brain creatine kinase (CKB)/beta-galactosidase fusion protein in Escherichia coli has allowed isolation of a rat CKB cDNA clone by direct antibody screening of a rat brain gamma gt11 expression library. This clone is 1416 bp long and includes 202 bp of 3'-untranslated region and 29 bp of 5'-untranslated region. The coding sequence of this clone has enabled us to deduce the complete amino acid (aa) sequence of rat CKB protein.

Amino Acid Sequence↗
PubMed1996

MB fraction of cumulative creatine kinase correlates with insulin secretion in patients with acute myocardial infarction: insulin as a possible determinant of myocardial MB creatine kinase.

To test whether insulin is a regulatory factor of myocardial MB creatine kinase content, we investigated the correlation between the ability of insulin secretion and the MB fraction of cumulative CK released in patients with acute myocardial infarction. We analyzed 18 patients who underwent successful direct angioplasty within 10 hours of the onset of their first myocardial infarction. Exclusion criteria were age more than 75 years, heart failure, severe obesity, multivessel disease, and history of diabetes mellitus. Cumulative activity of serum MB CK divided by that of total CK was defined as MB%, which was considered to represent myocardial MB CK content. Two weeks or more after the onset of myocardial infarction, 75 gm oral glucose tolerance test with serial determination of plasma glucose and serum insulin (0, 0.5, 1, 2, 3 hours) was done. Urinary and plasma catecholamines and echocardiographic left ventricular (LV) mass were measured. MB% significantly correlated with insulinogenic index (r = 0.564, p = 0.019), insulin area (r = 0.594, p = 0.012), insulin area/glucose area (r = 0.630, p = 0.007), and urinary adrenaline (r = -0.542, p = 0.025) and tended to correlate with plasma adrenaline (r = -0.431, p = 0.084). Age, body mass index, infarct size, glucose metabolism, and LV mass were not significant univariate predictors of MB%. Multivariate analysis showed that the ability of insulin secretion contributed to MB% more than catecholamines did and that insulin area/glucose area was the strongest independent predictor of MB% (t = 3.01, p = 0.015). Thus MB fraction of cumulative CK released, indicative of Myocardial MB CK distribution, strongly related to the ability of insulin secretion in subjects without overt insulin resistance. Regulation by insulin of myocardial MB CK is suggested.

Adrenergic Agonists↗
PubMed1996

Creatine kinase and creatine kinase-MB release after nontraumatic cardiac arrest.

The aim of the study was to describe the course of serum creatine kinase (CK) and its MB fraction (CK-MB) in patients surviving cardiac arrest, and to identify factors influencing CK and CK-MB release. The study was set in the community of Vienna, Austria. Data concerning cardiopulmonary resuscitation, collected within a period of 33 months, were evaluated retrospectively and compared with laboratory blood investigations collected prospectively (on admission and after 6, 12, and 24 hours) in 107 adult patients surviving a witnessed cardiac arrest for 24 hours. CK and CK-MB were elevated in >75% of the patients within 24 hours. Release of CK and CK-MB was mainly associated with electrocardiographic evidence of acute myocardial infarction (AMI) cumulative energy administered during defibrillation, and duration of chest trauma by compression. The CK-MB/CK ratio was elevated in 32% of the patients. Of patients with electrocardiographic evidence of AMI, only 49% had an elevated CK-MB/CK ratio. In conclusion, the elevation in serum CK and CK-MB fraction in patients after nontraumatic cardiac arrest is a frequent finding, and is associated with ischemic myocardial injury, as well as physical trauma to the chest. This should be considered when interpreting the course of CK and CK-MB fraction for the diagnosis of AMI.

Adult↗
PubMed1983

Changes in serum myoglobin, total creatine kinase, lactate dehydrogenase and creatine kinase MB levels in runners.

A comparison was made between pre- and post-run serum levels of total creatine kinase (CK), CK-MB isoenzyme, lactate dehydrogenase (LD) and myoglobin (MG) levels in 84 runners in each of three groups; male 5-10 mile run; female 5-10 mile run; and male 15-55 mile run. Levels of CK-MB and MG were quantified using radioimmunoassay, while total CK and LD were measured using spectrophotometric procedures. Post-run levels of all four markers were significantly higher than the pre-run levels (p less than 0.001). Although levels of enzymes and MG correlated with the sex and distance run, the post-run changes did not correlate with age nor with the smoking status of the runners. Elevation of these markers as a result of vigorous exercise in the absence of myocardial infarction requires cautious interpretation of serum enzyme changes in runners.

Adult↗
PubMed1994

Creatine kinase immunocytochemistry of human sperm-hemizona complexes: selective binding of sperm with mature creatine kinase-staining pattern.

OBJECTIVE: To examine the clinical significance of the increased sperm cytoplasmic content that is due to a fault of spermatogenesis, we have further studied the relationship between increased sperm creatine kinase (CK) concentrations and diminished fertilizing potential in men. In the present work, we used CK immunocytochemistry of human sperm-hemizona (HZ) complexes to examine whether the distribution of mature (clear heads), intermediate (sperm heads with light stippling), and immature (heads with heavy stippling or with solid CK staining) spermatozoa bound to the HZ would follow the incidence of these sperm in the samples tested, or if there is a preferential binding by the mature sperm. DESIGN: Two pairs of HZ were exposed to washed semen and to their swim-up sperm fractions. The sperm and sperm-HZ complexes were treated with a CK antibody followed by horseradish peroxidase immunostaining, and the sperm were evaluated for maturity. SETTING: Men presenting for fertility evaluation were studied in two university-based andrology laboratories. RESULTS: The binding of the HZ was selective for mature sperm as indicated by the incidence of intermediate and immature sperm in washed semen versus bound to the HZ (intermediate: 20.0% versus 1.4%; immature: 7.6% versus 0.5% [mean +/- SEM]) or in swim-up sperm fractions versus the HZ (intermediate: 18.7% versus 3.4%; immature: 2.5% versus 0.2%). The binding was almost exclusive to normal sperm (96.4% to 98.1%) whether the HZ were exposed to washed semen or swim-up fractions in spite of the five to ten times higher incidence of intermediate and immature sperm. CONCLUSIONS: Mature sperm selectively bind to the zona. We suggest that spermatozoa with immature CK-staining patterns are deficient in the site(s) of oocyte recognition and binding.

Blotting, Western↗
PubMed1999

Acute creatine ingestion in human: consequences on serum creatine and creatinine concentrations.

The aim of the study was to explore the effect of an acute dose of creatine (Cr) ingestion on serum Cr and serum creatinine (Crn) concentrations. Sixteen healthy subjects ingested a single dose of Cr (20 g) followed by the measurement of serum Cr and Crn concentration for 3 h up to a maximum of 6 h (n=6). In response to Cr ingestion a large rise in serum Cr concentration was observed (by 50 folds) occurring approximately 2 1/2h after the ingestion (peak value of 2.17 +/- 0.66 mmol x l(-1)). We also found a moderate but significant rise in serum Crn concentration averaging 13 % after 3 h (peak value at 99.5 +/- 10.5 micromol x l(-1)). A dose response curve obtained in two case studies, in whom different doses of Cr were ingested (0, 2.5, 5, 10, 15, 20 g and 0, 10, 20, 30 g), showed that serum Cr concentration as well as the peak time increased linearly with Cr ingestion. In addition, acute Crn ingestion (5 g) resulted in a substantial increase in serum Crn concentration (by 10 folds) but led to a minor rise in serum Cr concentration (by 2 folds). These results suggest that when acute doses of Cr are ingested in humans, the degree of conversion of exogenous Cr to Crn in the stomach and the gut can be considered as negligible following the first 6 h of ingestion. However, further studies are required to explore the prolonged effect of Cr on Crn metabolism.

Administration, Oral↗
PubMed2001

Creatine and the creatine transporter: a review.

The cellular role of creatine (Cr) and Cr phosphate (CrP) has been studied extensively in neural, cardiac and skeletal muscle. Several studies have demonstrated that alterations in the cellular total Cr (Cr + CrP) concentration in these tissues can produce marked functional and/or structural change. The primary aim of this review was to critically evaluate the literature that has examined the regulation of cellular total Cr content. In particular, the review focuses on the regulation of the activity and gene expression of the Cr transporter (CreaT), which is primarily responsible for cellular Cr uptake. Two CreaT genes (CreaT1 and CreaT2) have been identified and their chromosomal location and DNA sequencing have been completed. From these data, putative structures of the CreaT proteins have been formulated. Transcription products of the CreaT2 gene are expressed exclusively in the testes, whereas CreaT1 transcripts are found in a variety of tissues. Recent research has measured the expression of the CreaT1 protein in several tissues including neural, cardiac and skeletal muscle. There is very little information available about the factors regulating CreaT gene expression. There is some evidence that suggests the intracellular Cr concentration may be involved in the regulatory process but there is much more to learn before this process is understood. The activity of the CreaT protein is controlled by many factors. These include substrate concentration, transmembrane Na+ gradients, cellular location, and various hormones. It is also likely that transporter activity is influenced by its phosphorylation state and by its interaction with other plasma membrane proteins. The extent of CreaT protein glycosylation may vary within cells, the functional significance of which remains unclear.

Alternative Splicing↗
PubMed1987

Monoclonal antibody studies of creatine kinase. Antibody-binding sites in the N-terminal region of creatine kinase and effects of antibody on enzyme refolding.

(1) The binding sites of two monoclonal antibodies, CK-2A7 and CK-5H5, have been located to a 60-amino-acid sequence in the N-terminal region of creatine kinase (CK) by the use of chemical cleavage with formic acid (which cleaves proteins at Asp-Pro bonds) and cyanogen bromide (which cleaves at Met residues). (2) A simple method for preparing chemically-cleaved fragments of proteins for electrophoresis and Western blotting is described. (3) Binding studies with CK preparations from different animal species show that single amino acid changes at residues 39 or 82 prevent binding of CK-2A7 and CK-5H5 respectively. We suggest that Lys-39 and Glu-82 form parts of the binding sites on CK for the two monoclonal antibodies. The two sites lie in variable regions at each end of a highly-conserved sequence (residues 46 to 79) and are inaccessible to antibody in the native enzyme. (4) One of the antibodies, CK-2A7, inhibits the refolding of CK to native enzyme after denaturation by urea.

Amino Acid Sequence↗
PubMedDate not supplied

Creatine kinase and creatine kinase isoenzymes as a marker of uterine activity.

Creatine kinase (CK) and CK isoenzymes are known to fluctuate in labor. Reliable information about the longitudinal changes of CK and CK isoenzymes during labor is sparse. Nevertheless, they have been used to direct care in women with cardiopulmonary disease and preterm labor requiring tocolysis. This study evaluated fluctuations of CK and its isoenzymes longitudinally across labor in 49 women. Blood samples were obtained at 33 to 34 weeks' estimated gestational age, on admission in labor at 3 cm or less dilation, 8 cm to complete dilation, and postpartum in the recovery room. Specimens were analyzed for total CK, CK-MM, CK-MB, and CK-BB activity. CK levels increased for all peripartum patients (p < 0.001). CK activity at 3 cm was greater than at 34 weeks (p < 0.01). Furthermore, the early rise in CK activity was greater in those in active labor compared with those who required oxytocin stimulation (p < 0.001). CK values at 8 cm and postdelivery (mean IU/liter) were often above nonpregnant norms. The early rise of CK in spontaneously laboring patients versus those requiring oxytocin augmentation may represent a difference in uterine activity. Nonpregnant normative data for CK is not appropriate when assessing cardiovascular side effects of betamimetic therapy.

Biomarkers↗
PubMed2000

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart.

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1997

Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios.

Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.

Animals↗
PubMed2000

Is creatine kinase responsible for fatigue? Studies of isolated skeletal muscle deficient in creatine kinase.

Creatine kinase (CK) is a key enzyme for maintaining a constant ATP/ADP ratio during rapid energy turnover. To investigate the role of CK in skeletal muscle fatigue, we used isolated whole muscles and intact single fibers from CK-deficient mice (CK(-/-)). With high-intensity electrical stimulation, single fibers from CK(-/-) mice displayed a transient decrease in both tetanic free myoplasmic [Ca(2+)] ([Ca(2+)](i), measured with the fluorescent dye indo-1) and force that was not observed in wild-type fibers. With less intense, repeated tetanic stimulation single fibers and EDL muscles, both of which are fast-twitch, fatigued more slowly in CK(-/-) than in wild-type mice; on the other hand, the slow-twitch soleus muscle fatigued more rapidly in CK(-/-) mice. In single wild-type fibers, tetanic force decreased and [Ca(2+)](i) increased during the first 10 fatiguing tetani, but this was not observed in CK(-/-) fibers. Fatigue was not accompanied by phosphocreatine breakdown and accumulation of inorganic phosphate in CK(-/-) muscles. In conclusion, CK is important for avoiding fatigue at the onset of high-intensity stimulation. However, during more prolonged stimulation, CK may contribute to the fatigue process by increasing the myoplasmic concentration of inorganic phosphate.

Animals↗
PubMed2002

Role of creatine kinase in cardiac excitation-contraction coupling: studies in creatine kinase-deficient mice.

To understand the role of creatine kinase (CK) in cardiac excitation-contraction coupling, CK-deficient mice (CK-/-) were studied in vitro and in vivo. In skinned fibers, the kinetics of caffeine-induced release of Ca2+ was markedly slowed in CK-/- mice with a partial restoration when glycolytic substrates were added. These abnormalities were almost compensated for at the cellular level: the responses of Ca2+ transient and cell shortening to an increased pacing rate from 1 Hz to 4 Hz were normal with a normal post-rest potentiation of shortening. However, the post-rest potentiation of the Ca2+ transient was absent and the cellular contractile response to isoprenaline was decreased in CK-/- mice. In vivo, echocardiographically determined cardiac function was normal at rest but the response to isoprenaline was blunted in CK-/- mice. Previously described compensatory pathways (glycolytic pathway and closer sarcoplasmic reticulum-mitochondria interactions) allow a quasi-normal SR function in isolated cells and a normal basal in vivo ventricular function, but are not sufficient to cope with a large and rapid increase in energy demand produced by beta-adrenergic stimulation. This shows the specific role of CK in excitation-contraction coupling in cardiac muscle that cannot be compensated for by other pathways.

Adrenergic beta-Agonists↗
PubMed1978

Diagnostic value for acute myocardial infarction of creatine kinase and lactate dehydrogenase isoenzymes compared with total enzymes. Creatine kinase isoenzyme specificity for myocardial damage.

The diagnostic value of creatine kinase (CK) and lactate dehydrogenase isoenzymes was investigated in a prospective study of 201 patients with suspected acute myocardial infarction (AMI). The isoenzymes were analyzed with a column chromatographic method. The patients' final diagnoses were made according to the WHO criteria without knowledge of the isoenzyme results. The prevalence of AMI was 50%. The diagnoses were predicted with significantly greater reliability with the isoenzyme than with the total enzyme determinations in most of the patients. However, the greater diagnostic reliability had sufficient therapeutic consequence to justify the extra analytical cost only in patients for whom the diagnosis must be based mainly on the enzyme results. The CK isoenzyme specificity for myocardial damage was studied in populations with low prevalence of heart disease. In a group of 39 patients who had elevated total CK due to noncardiac disease there were five with elevated isoenzyme values, but since among 69 young healthy persons none had elevated isoenzymes, this was taken to indicate that the isoenzymes may be leaked into the blood from other organs than the heart.

Acute Disease↗
PubMed1978

Maternal, foetal and neonatal blood creatine-phosphokinase-activities and creatine-phosphokinase-isoenzymes after labour with and without epidural analgesia and after caesarean section.

Maternal, cord and neonatal blood creatine-phosphokinase (CK) activities and CK-isoenzymes were determined in connection with 24 labours after normal pregnancies. In eight cases there was a normal vaginal delivery; in another eight cases there was a normal vaginal delivery with segmental epidural analgesia; and in the remaining eight cases there was an elective caesarean section under general anaesthesia. The lowest maternal blood CK-activities were seen in connection with caesarean section. There was a statistically significant difference in the maternal CK levels after delivery between cases with caesarean section and normal labour (P less than 0.05) and between cases with caesarean section and labour with segmental epidural analgesia (P less than 0.001). Most of the maternal CK-activity was of muscular type, and there were no statistically significant differences in isoenzyme fractions between the different groups. The neonatal blood CK-activities were at their peak at the age of 1 day in all groups. Between the groups there were no statistically significant differences in cord or neonatal blood CK-activities at any time. Most of the cord and neonatal blood CK-activity was also of muscular type, and there were no significant differences in the CK-isoenzyme fractions between the groups.

Adult↗
PubMed1985

Myoglobin, creatine kinase and creatine kinase subunit-beta in serum from patients and relatives with hypokaliaemic familial periodic paralysis.

Increased levels of serum myoglobin (S-myoglobin) were found in 6 investigated patients with hypokaliaemic familial periodic paralysis (hypoKPP) in their habitual condition. Furthermore, in 3 of the 6, increased levels of serum creatine kinase (S-CK) were found. These findings support the hypothesis that hypoKPP is a permanent myopathy, which is aggravated during attacks. It was found that 1 definite carrier had elevated S-myoglobin and among healthy relatives 6/11 had elevated S-myoglobin and 3/11 elevated S-CK. The use of S-myoglobin and S-CK for carrier detection is discussed.

Adolescent↗