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1.
Creatine kinase (CK; EC 2.7.3.2) isoenzyme BB extracted from brains of rats reportedly undergoes modification at 37 degrees C, leaving an electrophoretic variant that accounts for most of the residual CK activity. This variant, called CK-BB', migrates on electrophoresis similarly to creatine kinase isoenzyme MB. Using electrophoresis and immunoinhibition with antiserum to creatine kinase isoenzyme MM, we found CK-BB to be the only identifiable cytoplasmic isoenzyme in surgical samples from human brain and intestine. In contrast, we found that some samples of brain obtained at autopsy contain CK-BB'. We also found that CK-BB extracted from human brain was converted to CK-BB' upon incubation in serum or plasma at 37 degrees C. We found a similar development of CK-BB' in incubation mixtures of serum or plasma containing CK-BB obtained from surgical samples of human intestine. The development of CK-BB' during infarction of the gastrointestinal system may thus be a source of false-positive CK-MB in the laboratory verification of myocardial infarction when electrophoresis is used as the only method to identify CK isoenzymes.  相似文献   

2.
This report describes the procedures for isolation of creatine kinase BB isoenzyme (CK-BB) from human placenta on preparative polyacrylamide gel electrophoresis. 2.5 mg of CK-BB was purified from a 100-g portion of the human placenta, which had a mean specific activity of 957 kU/g and a mean yield of 16%. The placenta CK-BB exhibited single protein bands on several electrophoretic techniques. In addition, both of the placenta and brain CK-BB preparations were individually iodinated and the identical immunological properties of both the CK-BB preparations were confirmed in radioimmunoassay.  相似文献   

3.
The goal of this work was to find out whether it is possible to measure all three creatine kinase isoenzymes under the same reaction conditions in spite of their different kinetic properties. We found the tightest substrate binding for purified human BB, followed by the MB And MM isoenzyme preparations for both creatine phosphate and ADP. An increase in substrate concentration usually resulted in an inhibition. Nevertheless, it was possible with a method optimized for the MM isoenzyme also to measure the BB and MB isoenzymes at a rate of inhibition of only 6 and 3%, respectively. Marked differences in the apparent Km values between purified and native MM isoenzyme in human serum may indicate that the enzyme declined in substrate affinity during the isolation procedure. The use of enzyme preparations for standardization purposes, therefore, is only suitable if their kinetic properties are close to those of the enzyme in serum. Difficulties in the calculation of the apparent Km values are discussed and the graphical procedures of Lineweaver and Burk and of Eisenthal and Cornish-Bowden compared.  相似文献   

4.
Total activities of creatine kinase (EC 2.7.3.2; CK) and lactate dehydrogenase (EC 1.1.1.27; LD) and their isoenzymes were estimated in serum and tissue samples from patients with stomach adenocarcinomas who were to undergo gastric resection. Total CK activity (U/g protein) appeared to be markedly decreased in neoplastic stomach tissue. CK-BB was the predominant isoenzyme in both neoplastic and normal stomach tissues; however, the CK-BB/total CK ratio was increased in adenocarcinoma tissue. Macro CK type 1 was found in two neoplastic tissues and macro CK type 2 in 11. LD4 and LD5 isoenzymes were predominant in gastric tissues, but LD5 and the LD5/LD1 ratio were higher in adenocarcinoma tissue. At 24 h before surgery, CK-BB was demonstrated in sera of all patients and CK-MB in 69%. The CK-BB probably originated from neoplastic stomach tissue, which contains high CK activity, with BB isoenzyme predominating. After gastrectomy, CK and LD isoenzymes in sera were markedly increased by 24 h postsurgery. These alterations were attributed to release from damaged tissue during gastric resection.  相似文献   

5.
This paper describes the purification of human creatine kinase BB with high specific activity (1,122 U/mg). The procedure used resulted in a protein yield of 5.4 mg (21% recovery) from 150 g of brain tissue. Two-dimensional electrophoresis and PAGE studies indicated that purified CK-BB might exist as native isoenzyme along with structural aggregates since the multi-banded appearance was reduced to a single band with sodium dodecyl sulfate treatment but not with 2-mercaptoethanol. Investigators are cautioned not to store brain tissue for prolonged periods of time before isolation of the isoenzyme as this may lead to protein redistribution with additional bands becoming evident on PAGE.  相似文献   

6.
Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2, CK) BB isoenzyme from stomach tumor tissue was partially purified and its characteristics were compared with those from healthy tissue. Molecular mass of tumor CK-BB was estimated to be 82 000 by polyacrylamide gel electrophoresis. Tumor CK-BB was separated into 2 main subbands around pH 4.5 and 11, minor subbands around pH 5-7.5 by agarose isoelectric focusing. The isoenzyme reacted with anti-human brain CK-BB antibodies and formed a hybrid, CK-MB, with CK-MM prepared from healthy human skeletal muscle. The above physicochemical and immunological characteristics of tumor CK-BB were the same as those of normal CK-BB from normal stomach tissue. Optimum pH of tumor CK-BB was more acidic than that of normal CK-BB. Affinity for creatine phosphate and heat sensitivity of tumor CK-BB were slightly lower than those of normal CK-BB. Tumor CK-BB was more stable after iodoacetamide and urea treatments.  相似文献   

7.
BACKGROUND: Serum creatine kinase-MB isoenzyme (CK-MB) is widely used as a marker of myocardial injury. We prepared recombinant human CK (r-hCK) MB isoenzyme and examined its potential for use as a control material for assay of CK-MB in serum. METHODS: cDNAs encoding CK-M and CK-B subunits were inserted into the same plasmid vector, followed by transformation of Escherichia coli. The resulting three types of CK isoenzymes were purified by conventional chromatography. RESULTS: The ratio of MB to MM to BB was 50:40:10 on the basis of CK activity. Highly purified CK-MB with a specific activity of 533 U/mg was produced in a yield of 5.7 mg/g of packed cells. Purified r-hCK-MB had the isoelectric point (pI 5.3) and molecular size (46 kDa for the subunit) of native CK-MB. Its immunoreactivity in an ELISA using antibody against native heart enzyme was similar to that of cardiac CK-MB. The r-hCK-MB retained >90% activity for at least 4 months at 11 degrees C in a delipidated serum matrix in a liquid form at a concentration of 118 U/L. CONCLUSIONS: r-hCK-MB shows key properties of the native cardiac isoenzyme and may be useful as a control and calibrator for serum assays of CK-MB.  相似文献   

8.
We report the case of a patient with persistently above-normal activity of creatine kinase (CK) in serum, a major fraction of which on electrophoresis moved as a band between the MM and MB isoenzymes and on anion-exchange column chromatography eluted in the MB fraction. Measurements in the presence of specific M or B subunit-inhibitory antibodies indicated that 93% of the activity consisted of B-isomers. From these experiments we conclude that the abnormal CK is of BB nature. Gel filtration and immunoglobulin precipitation showed that the CK-BB was complexed with IgG. Normal CK-BB, when mixed with the patient's serum, was converted to macro CK-BB. In vitro stability of 37 degrees C of the abnormal enzyme was much greater than that of normal BB and MM isoenzymes. Following this finding, we then assessed 310 sera, received for enzyme assay by the clinical laboratory, for electrophoretically abnormally migrating CK isoenzymes. Of these, five (1.6%) contained such enzymes, all being of BB nature. They were of increased molecular mass, and at least three of them were complexed with IgG.  相似文献   

9.
The MM, MB and BB isoenzymes of human creatine kinase (CK) were separated by elution from micro-columns of DEAE-Sephadex A-50 with Tris buffer containing increasing concentrations of NaCl at pH 7.0, instead of pH 8.0 as has commonly been used. Since pH 7.0 is close to the pH optimum of CK, this allowed the use of four times larger aliquots of the eluates for the estimation of CK activity and, consequently, a 4-fold increase in sensitivity. Using serum specimens from patients with acute myocardial infarction, there was a good correlation of the CK-MM (r = 0.99) and CK-MB (r = 0.93) activities obtained with the two buffer systems. Similarly, normal sera had CK-MB and CK-BB activities of less than 2 U/l with both buffer systems. Comparison of the composition of serum proteins in the eluates by conventional electrophoresis revealed that although the distribution of CK isoenzymes separated by the two buffer systems was similar, the distribution of proteins at pH 7.0 showed an appreciable shift of protein from the MB to the MM eluates.  相似文献   

10.
Antisera against the crystallized creatine kinase isoenzymes from human skeletal muscle (MM) and from human brain (BB) were produced in rabbits. Both the MM and BB isoenzymes were precipitated quantitatively by their homologous antisera. No cross-reaction was observed. The hybrid MB from human heart muscle could not be precipitated completely by either of the two antisera. In artifical mixtures the concentrations of individual creatine kinase isoenzymes were determined from the percentage of non-precipitable activity in the supernatant after reaction with each of the antisera.This immunotitration assay was applied to study the quantitative distribution of creatine kinase isoenzymes in extracts of human tissues. The isoenzyme patterns obtained were compared with those determined by electrophoretic analysis.In sera of patients with myocardial infarction, the immunotitration assay allowed the sensitive and rapid quantitation of creatine kinase isoenzymes, especially of the “infarct-specific” hybrid MB, even in sera with low total activity. This indicates that the method is of diagnostic value.  相似文献   

11.
We demonstrate that human placenta is a convenient source of creatine kinase isoenzyme BB. We compare the physicochemical and catalytic properties with those of other creatine kinase isoenzymes: purified human abdominal muscle MM and brain BB. We also describe a stabilizing medium for creatine kinase BB. Human placental and brain BB have similar catalytic properties, the respective Km values for creatine phosphate being 0.66 and 0.56 mmol/L and for adenosine diphosphate 89 and 70 nmol/L.  相似文献   

12.
G Lum 《Clinical chemistry》1979,25(6):873-876
The MB isoenzyme of creatine kinase (CK) may be prepared in vitro from rabbit serum containing only the MM and BB isoenzymes, by means of a hybridization technique. The MM and BB dimers dissociate in 4 mol/L urea, which allows random recombination of M and B monomers. A liquid CK-isoenzyme control can be made from mixtures of rabbit sera obtained after hybridization and stabilized with glycerol and 25 mmol of 2-mercaptoethanol per liter. A liquid control stored at 4 degrees C showed good stability over a three-month period, declining to a mean residual activity of CK of approximately 90% after three weeks and a mean residual activity of MM, MB, and BB of 80--85% after six weeks. At 25 degrees C, CK activity of the liquid control declined to 75--80% after the fourth week. CK-BB at 25 degrees C was the least stable isoenzyme, declining to 75% after the third week and reaching 60% of activity after 12 weeks. CK-MB and CK-MM showed approximately 10--15% less stability at 25 degrees C than at 4 degrees C.  相似文献   

13.
Creatine kinase isoenzymes in human cerebrospinal fluid and brain   总被引:1,自引:0,他引:1  
Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.  相似文献   

14.
In this "column-batch" method for separating the MB and BB isoenzymes of creatine kinase and the LD1 isoenzyme of lactate dehydrogenase, one can, alternatively, separate MB from BB or obtain a combined fraction containing MB, BB, and LD1. The principal advantage is that the resulting fractions are twofold as concentrated as was the applied sample. Thus, activity can be measured by conventional automated methods, with no need for the modifications to compensate for diluted fractions that are required by other ion-exchange methods. Another advantage is the total absence of interference by the MM isoenzyme. A strong anion exchanger (AG-MP1, Bio-Rad) is used in the acetate form at pH 6.3. There is no retention of MM; retained MB, BB, and LD1 are eluted with a solution of magnesium acetate. Results are compared with those obtained for subunit B and LD1 by immunoinhibition. Results with patients are considered consistent with myocardial infarction if MB exceeds 20 U/L and 3% of the total CK and LD1 exceeds 130 U/L or 28% of the total LD activity.  相似文献   

15.
BB isoenzyme of creatine phosphokinase (CK-BB) obtained from human brain-extract changes its electrophoretic mobility after incubation in human serum at 37° C. No change of electrophoretic mobility of CK-BB is observed after incubation in isotonic saline. We have shown by means of immunoprecipitation with specific antibodies that the structure of CK-BB is not changed. These findings are supported by other authors and make the diagnostic value of electrophoretic separation of CK isoenzymes doubtful as after a 3-h incubation CK-BB migrates similarly to CK-MB and consequently may be misinterpreted.  相似文献   

16.
This work describes the action of the lysosomal enzymes arylsulfatase A (EC 3.1.6.1) and sialidase (EC 3.2.1.18) on human creatine kinase (CK, EC 2.7.3.2) isoenzyme BB. The isoenzyme, which gives a positive reaction with the periodic acid-Schiff reagent, contains 12 molecules of sulfate and two molecules of sialic acid per molecule. On treatment with arylsulfatase, CK-BB lost enzyme activity but retained immunoreactivity, its isoelectric point was altered, and it was partly bound to a "Glyco-gel" affinity column. On treatment with sialidase, the isoenzyme lost activity, its immunoreactivity was decreased by 70%, and the inactivated CK-BB would not bind to either "Glyco-gel" or concanavalin A. We propose that the sulfate groups are involved in maintaining the integrity of the active site of the enzyme but are not involved in antigenic recognition sites on the molecule. Sialic acid plays an important role in both the structural pattern of the antigenic determinant and the active site of CK-BB.  相似文献   

17.
Creatine kinase BB isoenzyme (CK-BB) was detected in abnormal amounts in serum samples from 11 of 46 patients with Stage D carcinoma of the prostate by electrophoresis. Thirteen of 46 Stage D patients had elevated acid phosphatase values and 10 of these 13 had elevated CK-BB. CK-BB elevations were less frequent in earlier stages of prostatic cancer; Stage C: 0 of 35, Stage B: 1 of 26, Stage A: 0 of 3 and none in a group of 35 with BPH, prostatitis and bladder cancer. Results of CK-BB by a specific radioimmunoassay correlated well with those obtained by electrophoresis in most cases. Several patients were followed over time and data on CK-BB is presented for this interval. The origin of the CK-BB is still unclear. The BB isoenzyme predominates in prostatic tissue and CK-BB is the fetal form of the enzyme in human muscle and myocardium. The increase in serum CK-BB may be related to increased release of the isoenzyme, either from the prostate itself or from a metastatic lesion, or may represent a release of the fetal form of the enzyme from dedifferentiated tumor tissue.  相似文献   

18.
The activity of creatine kinase isoenzyme BB (CK-BB) in serum is rarely abnormally high (i.e., detectable). An increase in immunoreactive CK-BB or CK-BB activity in patients with prostatic disease has been proposed as an indication of prostatic adenocarcinoma. Here we report the case of an elderly man with massive benign prostatic hyperplasia but no clinical or pathological evidence of prostatic adenocarcinoma, whose serum CK-BB activity was found by agarose gel electrophoresis to be 1 U/L (normal: 0%), 10% of his total CK activity. Serum CK-BB activity was further increased to 16 U/L (20% of total CK activity) 1 h after prostatectomy, but became undetectable by the second day after the operation. The findings suggest that: (a) the source of the serum CK-BB activity was the enlarged prostate gland; (b) abnormally high CK-BB activity in serum of men with prostatic disease does not necessarily indicate the presence of prostatic adenocarcinoma; and (c) myocardial injury could be erroneously diagnosed postoperatively in prostatectomy patients if CK isoenzyme methods are used that do not consistently separate "heart-specific" CK-MB from CK-BB.  相似文献   

19.
Creatine kinase and its isoenzymes in neoplastic disease   总被引:1,自引:0,他引:1  
The CK-BB isoenzyme is ubiquitous in neoplastic tissue, but with low activity. Accordingly, it might be a nonspecific and insensitive tumor marker. Evaluation of BB isoenzyme in serum might indicate the extent of diseases or the response to therapy. The presence of CK-MB in patients with cancers may cause confusion with AMI. Serial determinations of both CK and lactate dehydrogenase isoenzymes are of great help in differential diagnosis. The presence of mit-CK is a poor prognostic sign in patients with malignancy. The greatest clinical significance of CK-BB and macro-CK isoenzyme lies in their effect on various assays for CK-MB. Macro-CK types 1 and 2 are much more heat stable than are CK-MB and CK-BB, and so by heating samples for 20 min at 45 degrees C the presence of thermostable macro types can be demonstrated. Macro-CK type 2 has a much higher activation energy than macro-CK type 1. If macro-CK is present, determination of the activation energy easily differentiates between types 1 and 2. CK-Bi seems to be glycosylated protein, and it is thought that glycosylation may be a general way of enzyme inactivation. If inactivation inside the cell is postulated, it has to be shown that enzymes indeed pass into the cell compartments where glycosylating enzymes are located. Another possible mechanism is within the circulation. Whether malignant cells themselves produce Ck-Bi or if inactivation occurs in the blood is still unknown. In this connection, one finding is that in plasma of cancer patients, CK-Bi can be reactivated to CK-BB by mercaptoethanol to 95%, whereas in plasma of normal persons there is no reactivation of the much lower CK-Bi concentrations.  相似文献   

20.
The method is based on the selective activating capacity of dithiothreitol on creatine kinase isoenzyme MB, after isoenzyme MM is activated by glutathione. Isolated isoenzymes MM and MB of human and canine origin were assayed individually and in mixtures of known activities. When glutathione was present in the assay medium the activity of each isoenzyme could be measured individually, but glutathione did not activate isoenzyme MB if it was present in a mixture with MM. Dithiothreitol, added to the serum before assay, activated the isoenzyme MB in the mixture. Values for MB activities obtained for isolated isoenzyme MB and for the isoenzyme mixture after dithiothreitol was added averaged 110 and 111 U/liter, respectively (r = 0.998; y = 1.007 x + 0.298; n = 10). In the serum of 40 patients with documented acute transmural myocardial infarction, the mean proportion of isoenzyme MB activity measured in this way was 5.5% (coefficient of variation, 7.7%). Isoenzyme MB activities measured by use of dithiothreitol compared well with those obtained by conventional electrophoresis/spectrophotometry (r = 0.998; y = 1.09x -0.65) and spectrofluorometry (r = 0.996; y = 1.10 x + 0.80). The assay of MB activity by the dithiothreitol method was automated, by use of an Abbott Bichromatic Analyser and a Calbiochem Super-Stat Pack Kit. In 60 isoenzyme MB determinations the manual and automated method correlated well (r = 0.990; y = 1.0x -1.36). The simplicity of isoenzyme MB determination by use of dithiothreitol and its ease of automation allow routine monitoring of the isoenzyme activity in patients with ischemic heart disease.  相似文献   

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