Activity of creatine kinase B subunit, as measured by an immunoinhibition assay, is persistently increased in certain healthy, asymptomatic adults, whose values for total CK activity are within normal limits. Serum samples from two such individuals were investigated by electrophoresis, heat inactivation, determination of activation energies, and immunosorption (on protein A). The results demonstrated the presence of a circulating complex of IgG and CK-BB, which has been described as macro CK, type I. To our knowledge, this is the first report of such macro CK isoenzymes in healthy individuals.

Creatine kinase isoenzyme BB from rat brain was incubated with serum, dialyzed serum, or plasma, with added carboxylic acids, thiols, buffers, or cations. It was found to be very unstable at 37 degrees C under all these conditions, being transformed to CK-BB', a form that migrates like CK-MB on agarose electrophoretic plates. This transformation is enhanced at alkaline pH and, especially, by Zn2+. CK-BB' is quite stable, and probably results from binding of cations to CK-BB, because the transformation is prevented by EDTA and citrate.

We developed standards for creatine kinase (CK; EC 2.7.3.2) assays by expressing human CK cDNAs in COS cells. Cells were transiently transfected with full-length cDNAs for CK subunits M and B, individually and in combination; and subsequently, high concentrations of CK activity were present in the cell lysate (1.2 U/mg protein). These proteins exhibited the characteristic isoenzyme-specific electrophoretic mobilities for CK MM and BB isoenzymes. We also produced subforms of CK MM and MB, identical to the modified CK variants produced in plasma after muscle or myocardial injury, by mutating the cDNA for the CK M subunit to delete the carboxy-terminal lysine residue. When this construct was cotransfected with the normal cDNAs for CK M and B, five electrophoretically distinct CK isoenzymes were detected by nondenaturing electrophoresis: MM3, MM2, MM1, MB2, and MB1. These proteins retained 100% of their activity after storage of the cell lysates -20 or 4 degrees C for 3 months.

Most vertebrates possess two genes for cytoplasmic creatine kinase (CK) coding for muscle (M-CK) and brain (B-CK) isoforms which assemble into homo-dimeric (MM, BB) and hetero-dimeric (MB) active enzymes. In mammals and birds, a significant fraction of MM-CK is bound to the myofibrillar M-line where it is thought to facilitate energy buffering and transport. Myofibrillar binding is mediated by major and minor lysine charge clamp motifs (K104/K115 [major] and K8/K24 [minor] in chicken M-CK) located in the N-terminal region [J. Cell Biol. 149 (2000) 1225]. We have obtained the cDNA and deduced amino acid sequences for cytoplasmic CKs from two hagfish, Myxine glutinosa and Eptatretus stoutii, non-vertebrate craniates, and the sequences for two cytoplasmic CKs from the lamprey Lampetra japonica, a jawless true vertebrate. All four cDNAs code for CKs consisting of approximately 380 residues. Phylogenetic analyses showed that the hagfish and lamprey CKs are coded for by genes which are clearly muscle type (M) creatine kinases. Two of these four M-CKs have the K104/K115-equivalent residues of the major myofibrillar binding region while the other two have the K115 equivalent but lack the corresponding K104 residue. All four M-CKs lack the K8/K24 equivalent elements of the minor myofibrillar binding region. Comparison of these sequences to corresponding sequences of cytoplasmic CKs from two protochordates (tunicate, amphioxus) and M- and B-CKs from true fish and above reveal a pattern of acquisition (and loss) of key lysine residues consistent with the physiological context in which these enzymes operate.

Fluorescence emission intensity changes with two different excitation wavelengths were used to measure the unfolding rate constants of different domains of muscle type creatine kinase (CK-MM) according to the heterogeneity of aromatic amino acid distributions in the crystal structure of CK-MM. The results were compared with those of brain type creatine kinase (CK-BB) and dithio-bis(succinimidyl propionate) cross-linked CK-MM. CK-BB differed greatly in its distribution of aromatic amino acids in each domain and the unfolding process of cross-linked CK-MM was not accompanied by the dissociation of the dimer. The N-terminal domain of CK-MM was shown to be well protected by subunit interaction during the unfolding of CK-MM in 4 M urea. Dissociating the CK dimer in high urea concentration >> or = 6 M) eliminated the subunit protection. Subunit interactions are also important in preserving secondary structure and forming contracted conformation at low urea concentration.

Research on the stabilizing properties of creatine kinase isozymes CK-BB, CK-MB, and CK-MM showed that minor alteration of their sequence and structure influenced their stability significantly. An analysis of the stability of the isozymes in storage after freeze drying indicates that creatine kinase isozymes are all in monomer form because of the loss of subunit interactions. Freeze-drying leads to the oxidization of CK-BB and rearrangement of CK-MB. There are also differences in the unfolding of the isozymes in urea. CK-BB and CK-MB are unfolded in lower urea concentrations than CK-MM. Differences in the thermal unfolding were also examined by differential scanning calorimetry. This paper discusses the potential biological significance of these results.

The growing mechanism of the chronic subdural hematoma has not fully understood yet, in spite of numerous studies about hematoma neomembranes. However, it is well known that the majority of the chronic subdural hematomas are well healed by a simple irrigation of hematoma. These facts suggested that the hematoma contents could have important growing factors of the chronic subdural hematoma. Thus, LDH and CK activities were estimated in 52 cases of hematoma contents and 15 cases of hematoma neomembranes in order to search growing factors, biochemically. Hematocrit and hemoglobin values in hematoma contents were also examined simultaneously. As a result, hematocrit and hemoglobin values in hematoma contents were gradually increased, these facts might be due to the concentration of hematoma contents. LDH and CK activities in hematoma contents were high around 60 days after the hematoma inducing head trauma, and these enzyme activities were not correlated with hematocrit value. In isozyme analysis of LDH and CK activities, LDH-1,2 and CK-MM showed high values but CK-BB, MB could not be recognized. These findings suggested that LDH activity in the hematoma contents were caused by hemolysis which had been reported to be a main cause, and CK activity might originate from muscular tissues. Therefore, author hypothesized that the CK activity in hematoma contents had originated from the neomembrane, since there was a good correlation between the mature stage of neomembrane and the high level of CK-MM, and the myofibroblast was found in neomembrane recently. CK-MM could be released from the myofibroblast in neomembrane. However, CK activity in hematoma neomembrane could not be recognized, biochemically nor immunohistochemically.(ABSTRACT TRUNCATED AT 250 WORDS)

The biological diagnosis of prostatic carcinoma in relation with benign prostatic hypertrophy is essentially realized by the evaluation of plasma PAP or medullar PAP, the increase of which rises to 70% of the cases. This evaluation contains also other biochemical markers such as CK-BB, glucose-6-phosphate dehydrogenase, LDH 5 or alkaline phosphatase. The elevation of urinary polyamines is also correlated with the evolution of carcinoma. Other markers have been recently described such as PSA, useful both by evaluation in serum and by its identification on biopsy in histopathology. This exploration could be completed by the evaluation of androgenic receptors and of circulating androgens.

Alterations of serum creatine kinase isoenzymes were observed in five cases of prostatic carcinoma. Creatine kinase isoenzyme BB was found in the serum of two of three cases with metastases. Its presence in serum does not seem to be related to acid phosphatase activity but seems associated with extension of the tumor to other tissues. Preliminary studies on effusions from patients with malignant and non-malignant prostates showed that CK-BB was detectable only in cytology positive effusions. This finding suggests that CK-BB may be a tumor product rather than a result of a host response. The observation of CK-BB in a significant percentage of patients (two of three) with metastatic carcinoma of the prostate is of interest and suggests that CK-BB isoenzymes may have some predictive value in following patients with malignant disease.

We have previously demonstrated that mouse skeletal tissue, rat bone as well as rat or human derived bone cells in culture, show a sex-specific response to gonadal steroids in stimulation of the specific activity of the BB isozyme of creatine kinase (CK). This response could be modified by manipulation of the endocrine environment during early postnatal development. Moreover, pretreatment with vitamin D up-regulated the sex-specific responsiveness and sensitivity to gonadal steroids. In the present study we examine the differentiation pattern into osteoblast-like cells using dexamethasone (DEX) and 1,25 dihydroxy vitamin D3 (1,25D) and their effect on the acquisition of responsiveness to gonadal steroids by the differentiated cells. Cultured femoral bone marrow in the presence of DEX or 1,25D or both, were examined for their response to gonadal steroids by measuring the specific activities of alkaline phosphatase (AP) and CK BB. The constitutive level of CK in both male- and female-derived bone cells was decreased by DEX, by 1,25D or by both, whereas the constitutive level of AP was increased by DEX while decreased by 1,25D or by both. Following incubation of the bone marrow cultures with DEX, treatment with estradiol 17beta (E2, 30 nM, 24 h) stimulated CK activity in female derived bone cells, with no effect of treatment with dihydrotestosterone (DHT, 300 nM). In contrast, in male derived bone cells, DHT but not E2 increased CK activity. This sex-specific response was also achieved upon culturing with 1,25D and was significantly augmented by culturing with both. No response to gonadal steroids was seen with undifferentiated bone marrow cells. All cultures responded to IGF-I when cultured with or without DEX and/or 1,25D but with no augmentation by 1,25D. Gonadal steroids increased AP to a much lesser extent; but enzyme activity decreased in the presence of 1,25D. IGF-I stimulated AP slightly with no effect of 1,25D. These findings suggest that manipulation of the hormonal milieu in early stages of differentiation sequence of osteoblast-like cells, determines the subsequent selective responsiveness of the developing bone tissue to gonadal steroids.

BACKGROUND

The aim of this study is to evaluate the diagnostic value of the enzyme creatine kinase (CK) in the cerebrospinal fluid (CSF) of children with meningitis.

METHODS

CSF samples were collected from seventy one children suspected of having meningitis. The levels of total CK, CK-BB, Glucose, total protein, WBC counts, and culture were determined in the CSF. The cutoff value for total CK in the CSF was defined as 18 U/L.

RESULTS

Three cases (4%) of bacterial meningitis and 11 cases (15%) of aseptic meningitis were confirmed by culture. The sensitivity and specificity of total CK CSF level alone to diagnose bacterial meningitis were found to be 33% and 91% respectively. The positive and negative predictive values were found to be 14% and 98% respectively. On the other hand, the sensitivity and specificity of total CK level in aseptic meningitis were found to be 40% and 98% respectively and the positive and negative predictive values were 86% and 94% respectively. The sensitivity and specificity of total protein and glucose in CSF were also calculated. Streptococcus pneumonia and homophiles influenza were the main types identified in our cases.

CONCLUSIONS

Measuring the total CK level in the CSF may be very useful in diagnosis of meningitis if only combined with other CSF markers. It is not of any much benefit if it is used solely.

We describe a new compound, N-dibenzylphospho-N'-3-(2,6-dichlorophenyl)-propylguanidine (DPPG), and our study of its interaction with cytosolic CK. To our knowledge, it is the most potent inhibitor known for CK: the Ki value versus ADP was 330 nM and 110 nM for CK-MM and BB respectively. In view of the inhibition pattern, Ki(app) dependencies on the second substrate, and very low Ki values, we conclude that DPPG binds to the active site as a bisubstrate-type analog. This bisubstrate analog confirms different mechanisms for CK-MM and BB: in spite of a more than 80% similarity in amino-acid sequences, both isoenzymes are random at pH 8.6 but CK-BB has an ordered mechanism at pH 6.6.

We report a case of a woman (58 years old) with persistent and elevated CK activities and without corresponding clinical signs. For seven months CK-B activity was determined above normal by an inhibition test. By exclusion chromatography, ion-exchange chromatography, electrophoresis of CK isoenzymes and immunological methods we were able to explain this phenomenon as Macro CK consisting of immunoglobulin G bound CK-BB.

We measured the BB isoenzyme of creatine kinase by a specific radioimmunoassay in the serum of 47 patients following cardiac surgery. A sharp increase in CK-BB occurred immediately after surgery, with rapid return to baseline by the fourth post-operative day. This data, along with other reports in the literature, suggest that CK-BB is released into the circulation following myocardial insult.

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 beta-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 less than 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.

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.

Creatine kinase (EC 2.7.3.2) BB isoenzyme (CK-BB) was purified to homogeneity from canine and human brain tissues. The purified protein from both sources exhibits Mr of 84,700 daltons. The canine isoenzyme exhibits several properties similar to human isoenzyme with respect to reactive and total thiol groups, UV spectra, isoelectric points and reaction kinetics. While both canine and human CK-BB isoenzymes are unstable compared to other CK isoenzymes, canine CK-BB is even less stable than the human enzyme, losing most of its activity within 20 h at 4 degrees C at pH 5.0. Addition of 2-mercaptoethanol does not prevent rapid loss of the enzyme activity. Increasing the pH to 9.0, however, increases the stability of both CK-BB isoenzymes. Agarose electrophoresis demonstrated the presence of MM as well as BB isoenzyme in various parts of brain tissues. BB was present at an activity of 90.8-93.3 U/mg and MM at 6.7-9.2 U/mg.

Creatine kinase brain isoenzyme (CK-BB) may be released into the serum after brain injury. Its concentration was measured serially in a group of 80 term infants at risk of perinatal hypoxic damage. Their neurodevelopmental outcome was furthermore assessed at 12 months of age. Our results are consistent with the hypothesis that infants with high CK-BB concentration, measured in serum with a new and very sensitive method for enzyme analysis, have more frequently poor short-term outcome (p less than 0.02). These data suggest that early CK-BB measurement may be clinically useful to identify newborns at high risk for neurologic or developmental sequelae.

The mass concentration of creatine kinase BB (CK-BB) isoenzyme was measured in serum of 365 healthy children and adults with a CK-BB specific radioimmunoassay. Eight separate age groups (newborns, 4 days, and 1, 8, 14, 25, 40 and 65 years) were examined and the reference intervals for these groups were estimated. The concentration of CK-BB generally decreased with increasing age, most of the changes taking place within the first year of life. Thus, the median value at age 1 year (9.2 micrograms/l) was 1/15th of that observed at birth, 1/2 of that observed at age 4 days, but four times higher than that observed in adults. A transient increase in CK-BB concentration was observed in boys 14 years old. At this age in contrast to at all other ages the values measured in boys were significantly (P less than 0.005) higher than the values measured in girls In 27 of the sera from cord blood we also examined the relationship between estimates of CK-BB mass and activity, the latter being estimated by immuno-inhibition technique. The two estimates were highly correlated (r = 0.98), 2 micrograms of enzyme mass roughly equivalent to 1 U of enzyme activity.

Monoclonal antibodies were generated against rat uterine estrogen-induced protein--creatine kinase (CK-EIP)--and two (MAb-28 and MAb-78) were studied. These antibodies were IgM but differed in their complementary antigenic determinants both of which were detectable on denatured but not on native CK-EIP. MAb-28 reacted with other CK-BBs but not with CK-MMs whereas MAb-78 reacted with both types of CKs. A measurement of antigenicity with the monoclonal antibodies under calibrated conditions showed differences among the CKs, notably between CK-BB from rat brain and CK-EIP when both were probed with MAb-28. The antigenicity of CK-BB (rat brain) was significantly lower than that of CK-EIP, indicating that the former either expresses less copies of the determinant recognized by MAb-28 than CK-EIP does, or possesses a determinant which interacts with the antibody with lower affinity. The monoclonal antibodies should help elucidate structure-function relationships in CK-BB and CK-EIP molecules, their anatomic distribution and their physiologic, pathologic and experimental variations in relation to gene expression induced by sex hormones.

Creatine kinase (CK) and lactate dehydrogenase (LD) enzyme activities and isoenzymes were determined for synovial fluid, synovial membrane, and articular cartilage from 24 clinically normal equine tarsocrural (tibiotarsal) and femoropatellar joints. All 3 tissues contained LD isoenzymes LD1 to LD5, and CK isoenzymes BB and MM. The CK isoenzyme MB was not found. The similarities in isoenzyme composition of these 3 tissues made differentiation of the source of LD and CK impossible by isoenzyme pattern alone. Reference values for the total enzyme activities of specific joint tissues also had wide variations. The wide variation in activities, as determined by the enzymatic analysis of synovial fluid and a lack of tissue specificity in clinically normal equine joint tissue, indicated that those values were not predictive for the extent and type of tissue damage in equine joint disease. This hypothesis was confirmed when synovial fluids from 22 abnormal joints were analyzed for LD isoenzymes and total enzyme activity. The various causes of the joint problems were not distinguishable.

The studies regarding the effect of sodium dodecyl sulfate (SDS) on enzyme activities and structures can provide a valuable insight into public health. We have predicted the 3D structure of the brain creatine kinase (CK-BB) with a high resolution and simulated the docking between CK-BB and SDS. The predicted structure had a root mean square deviation of 0.51 A. The docking between CK-BB and SDS was successful with significant scores (-4.67 kcal/mol, AutoDock4 and -48.32 kcal/mol, DOCKCK-BB's folding behaviors. The two-phase rate constants as a first-order reaction were measured during inactivation. SDS strongly inhibited the CK-BB activity in a noncompetitive inhibition manner (K (i) = 1.22 mM). The tertiary structural change was induced by SDS binding with the exposure of hydrophobic surface. The methyl-beta-cyclodextrin was used to strip SDS from the enzyme molecule to reactivate. The changes of thermodynamic parameters for the SDS ligand binding such as enthalpy, Gibbs free energy, and entropy were obtained as -13 + or - 7.0 MJ/mol, 8.39 kJ/mol, and -42.754 kJ/(K mol), respectively. Our study provides important structural information for CK-BB and its interaction with SDS with an insight on its folding and inhibition kinetics.

Creatine-Kinase-BB (CK-BB) is a brain specific enzyme, with a prognostic value for the patient's outcome after head-injury. We have investigated 76 brain injured patients and attempted to show a correlation of the concentrations of CK-BB and the Glasgow-Outcome-Scale (GOS). Patients with a CK-BB concentration of more than 50 ng/ml died. Patients, who had a CK-BB concentration less than 25 ng/ml showed only minimal neurological deficits. Intracerebral contusions and acute subdural haematomas showed the highest CK-BB concentration, indicating a high degree of braintissue-damage. CK-BB seems to have no correlation with the age of patients. Normalisation of elevated CK-BB levels do not correlate with recovery from neurological deficit.

Five monoclonal antibodies (CKM-B07, F12, D08, H09 and G01) against porcine creatine kinase (CK; EC 2.7.3.2) MM isoenzyme, which inhibit the enzymatic activity, were prepared. The hybridomas which produced monoclonal antibodies were screened by direct measurement of the inhibitory activity of their culture supernatant. Only two of them, however, were found to be measurable by an enzyme-linked immunosorbent assay with porcine CK-MM as an antigen. CKM-G01 inhibited 100% porcine CK-MM activity, while the others, 73-87%. On the other hand, only CKM-H09 inhibited porcine CK-BB activity (15%). CKM-F12 and D08 inhibited more than 50% CK-MB activity, whereas they did not inhibit CK-BB activity. The monoclonal antibodies were also tested for bovine, rabbit and human CK-MM. All the antibodies inhibited bovine and human CK-MM activity as well. In particular, CKM-G01 was found to exhibit more than 98% inhibition of all CK-MM activity tested, indicating that a common or very similar epitope which affects the activity is present on these enzymes. Admixing of CKM-B07 with other antibodies effected synergisms in inhibition, not only to porcine CK-MM activity but also to human CK-MM activity. A mixture of CK-B07 and G01 inhibited 100% human CK-MM activity, suggesting applicability of these monoclonal antibodies to clinical laboratory diagnosis.