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1.
Adenylate kinase (EC 2.7.4.3) interferes positively in the serum creatine kinase (EC 2.7.3.2) assay when the rate of ATP production is monitored by a coupled enzyme system. A dual assay, measuring creatine kinase and adenylate kinase activity, was used to evaluate AMP and other possible adenylate kinase inhibitors that would permit specific measurement of creatine kinase activity in the presence of adenylate kinase. We found that AMP, routinely included in the creatine kinase assay system to inhibit adenylate kinase, partially inhibits both human serum creatine kinase and purified creatine kinase from rabbit muscle. The amount of creatine kinase inhibition is related directly to the AMP concentration and inversely to the substrate (ADP) concentration. We found that 25 mmol/liter of fluoride inhibits adenylate kinase without measurable effect on creatine kinase activity. We developed a serum creatine kinase assay including fluoride, and compared it with the dual assay system and with two commercial assay kits. Other halides or adenosine 2'-monophosphate did not selectively inhibit adenylate kinase. 相似文献
2.
Interference of adenylate kinase with Oliver's method [Biochem. J. 61, 116 (1955)] for creatine kinase is usually suppressed by including an adenylate kinase inhibitor, AMP. We studied the kinetics and compared the inhibition capacities of AMP and diadenosine pentaphosphate. Both are competitive inhibitors, AMP being markedly weaker, with a Ki of about 300 mumol/liter for adenylate kinase from erythrocyte, muscle, and liver. AMP also weakly inhibitis creatine kinase. Diadenosine pentaphosphate inhibits erythrocyte and muscle adenylate kinase strongly (Ki about 0.03 mumol/liter), the liver isoenzyme less strongly (Ki about 3 mumol/liter), and has no effect on creatine kinase up to 100 mumol/liter. All three adenylate kinases may be present in a patinet's serum, causing sample blanks to be high in a creatine kinase assay that lacks inhibitors. In acute hepatic damage, liver adenylate kinase activity in serum can be grossly increased. Use of sufficient diadenosine pentaphosphate alone for complete inhibition is relatively expensive. Consequently, we recommend a combination of both inhibitors. Diadenosine pentaphosphate, 10 mumol, combined with 5 mmol of AMP per liter inhibits adenylate kinase from erythrocytes and muscle by 97% and from liver by 95%. 相似文献
3.
Elemental sulfur is a specific and potent inhibitor of the muscle-type isoenzyme of adenylate kinase (EC 2.7.4.3). We find inhibition by sulfur and by diadenosine pentaphosphate to be similarly potent and specific. Some properties of inhibition of adenylate kinase isoenzymes by sulfur are given. The adenylate kinase isoenzymes from skeletal muscle, brain, and heart muscle are inhibited by sulfur; those from liver and kidney are not. Other enzymes not inhibited by sulfur include the isoenzymes of creatine kinase (EC 2.7.3.2). We show that creatine kinase can be measured in serum when adenylate kinase is inhibited by sulfur, and that the sensitivity and specificity of this inhibition are of the same order as the inhibition of serum adenylate kinase activity by AMP plus diadenosine pentaphosphate. 相似文献
4.
L G Morin 《Clinical chemistry》1977,23(9):1569-1575
I have re-examined optimum reaction conditions for measurement of creatine kinase (EC 2.7.3.2). The optimum pH is 6.45, and 2,2-bis(hydroxymethyl)-2,2',2'-nitrotriethanol acetate, 200 mmol/liter, is the buffer of choice. Thioglycerol, 20 mmol/liter, is superior for both in-assay reactivation and for storage stability of sera. Fluoride, 25 mmol/liter, a broad inactivator of adenylate kinase (EC 2.7.4.3), has little effect on creatine kinase and is superior to AMP for adenylate kinase inhibition in the assay of creatine kinase. Magnesium ion, ADP, and buffer concentrations are interdependent and their optima must be determined together. The hexokinase/glucose-6-phosphate dehydrogenase activity ratio should not exceed 1.6. The range of linearity is limited by the glucose-6-phosphate dehydrogenase and NAD+ concentrations. Glucose-6-phosphate dehydrogenase, ADP, and NAD+ are the constituents most likely to result in unacceptable blanks. Creatine kinase is inhibited noncompetitively by anions: acetate and fluoride inhibit slightly, but sulfates, nitrates, and excessive chlorides should be avoided. 相似文献
5.
In search of an appropriate inhibitor to suppress the interference of adenylate kinase with the creatine kinase assay, we found that the combination diadenosine pentaphosphate (10 mumol/liter) and AMP (5 mmol/liter) is a better inhibitor than is fluoride (25 mmol/liter). The latter inhibits adenylate kinase uncompetitively and weakly (Ki = 2.5 mmol/liter), and must be incorporated in the starting reagent, and at 30 degrees C it becoms fully effective only after a lag phase of 6 min. In this concentration, fluoride inhibits adenylate kinase from erythrocytes, muscle, liver or platelets by 94, 92, 88, and 87%, respectively, and creatine kinase by 8%. Bromide and chloride also inhibit creatine kinase. Attempts to replace AMP by a specific inhibitor of liver adenylate kinase failed. Homologs of diadenosine pentaphosphate with either fewer or more phosphoryl groups in the polyphosphate bridge inhibited even more weakly than did the pentaphosphate. Platelets can significantly contribute to adenylate kinase activity in serum. The inhibitor combination inhibited adenylate kinase from platelets by 90%. 相似文献
6.
To establish optimum conditions for creatine kinase (EC 2.7.3.2) activity measurement with the creatine phosphate in equilibrium creatine reaction, we re-examined all kinetics factors relevant to an optimal and standardized enzyme assay at 30 and 25 degrees C. We determined the pH optimum in vaious buffers, considering the effect of the type and concentration of the buffer, as well as the influence of various buffer anions on the activity. The relation between activity and substrate concentration was shown and the apparent Michaelis constants of creatine kinase for creatine phosphate and ADP were evaluated. We tested the effect on creatine kinase measurement of the concentration of substrates (glucose and NADP+) in the auxillary and indicator reactions, especially the influence of the added auxiliary (hexokinase) and indicator (glucose-6-phosphate dehydrogenase) enzymes on the lag phase, at different temperatures. The NADP+ concentration proved to be the factor limiting the duration of constant reaction rate. We studied the inhibition of creatine kinase and adenylate kinase by AMP and established a convenient AMP concentration. For reactivation of creatine kinase, N-acetyl cysteine as sulfhydryl compound was introduced. Finally, we examined the relationship between activity and temperature. 相似文献
7.
The current methods for the determination of creatine kinase (EC 2.7.3.2) activity are derived from Oliver's method, in which AMP is used to decrease interference by adenylate kinase (EC 2.7.4.3). Recently, Szasz et al. and Rosano et al. described methods in which diadenosine pentaphosphate and fluoride, respectively, are used to reduce this interference. However, diadenosine pentaphosphate does not sufficiently inhibit such activity of hepatic origin, while fluoride alone can only inhibit it at concentrations at which the fluoride tends to precipitate as MgF2. Finally, Szasz et al., the Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical Physiology, and the German Society for Clinical Chemistry have proposed methods in which both AMP and diadenosine pentaphosphate are used to inhibit adenylate kinase. We have found that by using low concentrations of AMP and fluoride together, we can greatly diminish this interference without significant loss of creatine kinase activity and with no precipitation of MgF2. 相似文献
8.
We measured creatine kinase (EC 2.7.3.2) activity in 1009 serum samples from 538 patients in the intensive-care units of the University of Texas Medical Branch hospitals. Creatine kinase isoenzymes migrating cathodal to skeletal muscle creatine kinase (CK-MM) on cellulose acetate electrophoresis were found in sera from 14 of the 538 patients. Creatine kinase, lactate dehydrogenase (EC 1.1.1.27), aspartate aminotransferase (EC 2.6.1.1), and alanine aminotransferase (EC 2.6.1.2) activities were abnormally increased in these 14 patients. Liver lactate dehydrogenase isoenzyme (LDH5) and cardiac creatine kinase isoenzyme (CK-MB) were abnormally increased in 12 and eight of these patients, respectively. Ten of the 14 patients died during their hospital admission. We believe the creatine kinase isoenzymes that migrated cathodal to skeletal muscle creatine kinase (CK-MM) were of mitochondrial origin. 相似文献
9.
Sensitive assay of creatine kinase isoenzymes in human serum using M subunit inhibiting antibody and firefly luciferase 总被引:3,自引:0,他引:3
A Lundin I Styrélius 《Clinica chimica acta; international journal of clinical chemistry》1978,87(2):199-209
A sensitive method for the assay of B subunit and total creatine kinase activity is described. The ATP formation in the creatine kinase reaction is continuously monitored by measuring the bioluminescence obtained with a purified firefly luciferase reagent. The B subunit activity is determined using an M subunit inhibiting antibody resulting in greater than 99.5% and approximately 50% inhibition of MM and MB isoenzymes, respectively. The bioluminescent method correlated well with a similar spectrophotometric method in the assay of B subunit as well as total creatine kinase activity (r greater than or equal to 0.98). However, the bioluminescent assay is considerably more sensitive, allowing the assay of B subunit activity in serum from healthy individuals. This is due to the inherent sensitivity of the bioluminescent assay of ATP, a reduced analytical interference from adenylate kinase and a reduced reagent blank. The within-series precision at 1 U/liter and greater than 52 U/liter corresponded to a C.V. of 14% and 3%, respectively. The method is as rapid and suitable for routine work as spectrophotometric methods. From a clinical point of view the new method is of particular interest in the early diagnosis of small acute myocardial infarctions. 相似文献
10.
Paul R. Desjardins 《Clinica chimica acta; international journal of clinical chemistry》1982,121(1):67-78
The 600 × g paniculate fraction, obtained from the homogenates of human heart muscle, contained large quantities of an atypical creatine kinase (CK-Z). Creatine kinase Z migrated cathodically relative to CK-MM on agarose gel electrophoresis, and was not inhibited by antibodies directed against human CK-MM and CK-BB. Creatine kinase Z had an apparent Km for Mg-ADP and creatine phosphate of 0.04 mmol/1 and 1.3 mmol/1, respectively. This enzyme existed in two molecular forms; one form of molecular weight 33000–38000 in the presence of a buffer containing Tris-HCl (0.05 mol/1), EDTA (0.001 mol/1), and 2-mercaptoethanol (0.010 mol/1), pH 8.0; and another form having a molecular weight of 62000–68000 in the presence of a buffer containing sodium phosphate (0.020 mol/1) and EDTA (0.001 mol/1), pH 8.0.Creatine kinase Z had biochemical properties which were different from those of the other soluble creatine kinase isoenzymes (MM, MB and BB), but similar to those reported for mitochondrial creatine kinases isolated from other animal tissues. 相似文献
11.
S. Yamuna K. Valmikinathan D. Burt A.E.H. Emery 《Clinica chimica acta; international journal of clinical chemistry》1977,79(1):277-279
Independent studies by two different groups (Madras and Edinburgh) have failed to confirm the suggestion that measurement of the serum level of pyruvate kinase (EC 2.7.1.40, PK) may be superior to measurement of the serum level of creatine kinase (EC 2.7.3.2, CK) for detecting female carriers of X-linked Duchenne muscular dystrophy (DMD). At present the serum level of creatine kinase remains the best test for this purpose. 相似文献
12.
I Docherty J S Harrop K R Hine M R Hopton H L Matthews C J Taylor 《Clinical chemistry》1984,30(1):42-45
Changes in values for myoglobin, total creatine kinase (EC 2.7.3.2), and creatine kinase B-subunit in the serum of patients with thyroid disease are compared with values for these during the 24-h after myocardial infarction. Concentrations of all three of these muscle-derived proteins were significantly higher than normal in patients with primary hypothyroidism, and declined with treatment. Values for total creatine kinase activity were below-normal in hyperthyroid patients, but increased after treatment. Values for total creatine kinase and, to a lesser extent, myoglobin in hypothyroidism extend into the range of values observed after myocardial infarction. The mechanism of the changes in these analytes in hypothyroidism may be related to increased leakage from skeletal-muscle cells or diminished clearance from the circulation, or both. 相似文献
13.
In the recently published results of the 1977 College of American Pathologists Enzymology Survey, Some anomalous results were obtained in measuring the creatine kinase (EC 2.7.3.2) activity of the samples. The cause of these anomalies was discovered to be an arginine kinase (EC 2.7.3.3) contaminant in one of the enzyme pools used to make the samples. The contaminant shows cross reactivity with creatine phosphate and is activated by dithioerythritol but not the other commonly used thiol activators. 相似文献
14.
K. Adriaenssens G. Vermeiren 《Clinica chimica acta; international journal of clinical chemistry》1980,105(1):99-103
By increasing diadenosine pentaphosphate to a final concentration of 75 μmol to 100 μmol/l to inhibit excess adenylate kinase, a highly sensitive substrate in a gelatin matrix allowed the development of a simple and cheap electrophoretic technique for creatine kinase MM isozyme in dried blood samples. The method is suitable for neonatal Duchenne muscular dystrophy screening, and it is an alternative method for a previously described bio-luminescent assay, which requires special equipment. 相似文献
15.
We examined data on adenylate kinase (EC 2.7.4.3) activity and other clinical chemical values from patients with renal transplants by analysis of variance and discriminant analysis, using various combinations of variables in an attempt to find a predictor of transplant rejection. Some typical data are presented. We conclude that the combination of urinary adenylate kinase and creatinine clearance is the best predictor for identifying patients with transient or destructive renal transplant crises. 相似文献
16.
K Emancipator A P Kudelka G Bradford K A Leonard M H Zarrabi 《Clinical chemistry》1988,34(12):2600-2602
A 37-year-old man with metastatic immature (malignant) teratoma with prominent rhabdomyosarcomatous elements had markedly increased activity of creatine kinase (EC 2.7.3.2) MB in serum. There was no electrocardiographic evidence of infarction or ischemia, and autopsy revealed no myocardial infarction, significant coronary atherosclerosis, myocarditis, or invasion of the heart by tumor. A high proportion of the creatine kinase activity in a homogenate of the tumor was attributable to the MB isoenzyme. Persistent increases of creatine kinase-MB and an unusually high MB isoenzyme activity, out of proportion to total creatine kinase activity, may indicate a nonmyocardial origin of this isoenzyme. 相似文献
17.
Ian Smith W.H.S. Thomson 《Clinica chimica acta; international journal of clinical chemistry》1977,78(3):439-451
Allosterism allows individual assay of both isoenzymes, one abundant in muscle, of pyruvate kinase (PK), recently reported superior to serum creatine phosphokinase (CPK) in detecting patients with and female carriers of X-linked recessive (Duchenne) muscular dystrophy (DMD). Extensive comparative studies did not support these findings, and confirmed the marked superiority of CPK over variants of PK or other enzymes in sensitivity, stability and convenience.Deducting the adenylate kinase increment (AKI) further refined the CPK assay, eliminating the effect of haemolysis in diagnosis and enabling studies of blood cell content. Both leucocytes and erythrocytes liberated PK and lactate dehydrogenase (LDH) after brief chilling or disruption. Only erythrocytes showed a CPK content, however, constantly adjusted to match that of serum as if by free cell membrane passage, but less accomodating to a sudden large influx of CPK than of LDH, where an apparent buffering effect could account for differences in clinical response. 相似文献
18.
W Stein J Bohner M Eggstein H Lang 《Zeitschrift für klinische Chemie und klinische Biochemie》1983,21(12):859-876
It was the aim of the workshop to summarize present knowledge of the variant creatine kinases in human blood. Discussion was centered on the nature, measurement, and clinical significance of these variants. On the basis of the presented results, the different creatine kinase variants can be classified as follows: Normal size variants (80000 Daltons). These originate from postsynthetic modifications of the three dimeric isoenzymes creatine kinase-MM, MB or BB. The M subunit can be transformed by a serum constituent and these modifications result in at least three different creatine kinase-MM and two creatine kinase-MB variants, which still show catalytic activity. The mechanism of the postsynthetic alterations of creatine kinase-BB seems to be more complex: In vitro incubation of this isoenzyme even in a non-serum matrix changes its electrophoretic mobility and decreases activity. In vivo there is evidence that on the one hand intact creatine kinase-BB molecules are directly removed from the circulation. On the other hand, however, inactive creatine kinase-BB-protein is reported to occur in serum. Variants with higher molecular weights (greater than 200000 Daltons). These are termed macro creatine kinases. Macro creatine kinase type 1 comprises the immunoglobulin-bound creatine kinase isoenzymes. Of these immune complexes, IgG-linked creatine kinase-BB is well known and has been described in detail, whereas the occurrence of creatine kinase-MM-containing macro creatine kinases is still questionable. Macro creatine kinase type 1 is found in the blood of about 2% of all hospitalized patients. It occurs mainly in elderly women, but it is not known whether it has any special clinical significance. Macro creatine kinase type 2 is the term for an oligomeric form of mitochondrial creatine kinase released after breakdown of mitochondria in liver, heart and some tumours. After treatment with urea this oligomeric form is converted into a normal sized, dimeric creatine kinase-MiMi. Macro creatine kinase type 2 is found exclusively in seriously ill patients, and may occur in more than 3% of all hospitalized patients. This classification of the creatine kinase variants seems logical, since it takes into account the nature of the creatine kinase variants, and it permits the classification of all atypical creatine kinases described in the literature. As quantification and differentiation now become possible, further experimental and clinical investigations should provide the information necessary for a better understanding of the physiology and pathobiochemistry of these multiple forms of creatine kinase. 相似文献
19.
Creatine kinase (EC 2.7.3.2) B-subunit activity in serum may be routinely measured as residual activity after specific immunoinhibition of the M-subunit. We assessed the inhibition kinetics, specificity, completeness of inhibition, and inhibitory capacity of three different anti-M preparations, with use of isolated human BB, MM, and MB isoenzymes. The Scandinavian-recommended reaction system was used. We suggest a set of tentative quality requirements for anti-M for use in diagnosing acute myocardial infarction. The need to measure and subtract sample residual adenylate kinase activity was demosntrated. We describe a routine photometric method for determining B-subunit activity in serum. With the Scandinavian CK method the upper reference value for total creatine kinase in serum was found to be 150 U/L for women, 270 U/L for men. By bioluminescence, we found the upper reference value for B-subunit activity to be 6 U/L for both sexes. We discuss three different modes for applying B-subunit determinations to the diagnosis of acute myocardial infarction. 相似文献
20.
In this case of mixed small cell--large cell cancer of the lung in an elderly woman, creatine kinase (EC 2.7.3.2) isoenzymes were assayed serially because of chest pain. The proportions of serum CK-BB and CK-MB isoenzyme activities were persistently above normal (CK-MB 10-18%, normal less than 5%). Electrocardiograms revealed no signs of ischemia or infarction. At autopsy no gross or microscopic infarction or inflammation of the heart was seen. There was also no infarction of smooth or skeletal muscle. The tumor was the probable source of most of the circulating CK-MB isoenzyme. Future cases may pose a similar diagnostic dilemma: differentiating creatine kinase that is present as a result of myocardial infarction from tumor-related CK-MB. Whether or not CK-MB assay could be useful in detecting tumors remains to be investigated. 相似文献