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1.
Creatine Phosphokinase (CPK) in striated muscles shows only small changes in activity before birth. After birth and during the first month of extrauterine life the activity increases rapidly. The largest increase is seen in muscles with a glycolytic energy metabolism (m. long, dorsi) and the smallest in muscles with an oxydative energy metabolism (m. flexor dig. ped. sup.). The differences between these groups of muscles are statistically significant. In heart tissue the increase in CPK activity is lower, the levels amounting to 40 to 47 % of those in striated muscles. Early in fetal life only the BB isoenzyme is found in striated muscles. Synthesis of M subunits of GPK starts between day 76 and 65 before birth and increases rapidly after this time leading to disappearance of the BB isoenzyme 24 days prior to birth and of the MB isoenzyme at birth. In muscles with an oxydative as well as in muscles with a glycolytic metabolism all GPK activity after birth is caused by the MM isoenzyme. All three isoenzymes are present in heart tissue at the earliest prenatal stage investigated, the pattern being dominated by the BB isoenzyme. During further differentiation the MM isoenzyme increases and the BB isoenzyme decreases. The development is completed during the first month after birth with a final isoenzyme composition of 81 % MM and 19 % MB isoenzyme. kw|Keywords|k]pigs; k]ontogenesis; k]creatine phosphokinase; k]activity; k]isoenzymes  相似文献   

2.
Investigation of creatine kinase isoenzyme activity in several cloned myogenic cell lines showed differences in B-type subunit expression. In cultures of myoblasts isolated from rat skeletal muscle by selective cell plating and in the cell lines M58 and M41, the activity of the mononucleated cells was of the BB isoenzyme. After cell fusion, MM, MB, and BB isoenzymes were present; the main activity was of the MM isoenzyme. In the myogenic lines L8 and L84, in cultures of mononucleated cells, creatine kinase activity was absent or barely detectable. The high creatine kinase activity after cell fusion was of the MM type. No BB and MB activity was detected in these lines at any stage of differentiation. The difference in expression of creatine kinase isoenzymes seems not to affect the expression of other parameters of differentiation.  相似文献   

3.
F Morel-Deletraz 《Enzyme》1979,24(4):277-279
Creatine kinase isoenzymes (MM, MB and BB) were isolated from human tissue by ion-exchange chromatography. The B subunit was found to be more heat sensitive than the M subunit. BB and MB isoenzymes respond similarly to heat inactivation. Our results are in contrast with the body temperature inactivation of the brain isoenzyme reported by Lindsey and Diamond.  相似文献   

4.
A bound form of creatine kinase associated with brain particulate was characterized by isoelectric focusing, antigenicity and chromatography and compared to muscle (MM), brain (BB), and heart mitochondrial isoenzymes. On partial purification and isoelectric focusing, the solubilized enzyme has a pl of 7.3, similar to the pl of muscle creatine kinase MM, pl 6.8, but different from brain creatine kinase BB, which precipitates on isoelectric focusing in sucrose or glycerol stabilized media at its calculated pl of 5.6. Gel filtration chromatography of deoxycholate solubilized particulate creatine kinase on Sephadex Gl50 reveals an estimated molecular weight of approximately 80,000 daltons. The brain particulate enzyme is antigenically distinct from both muscle and rat heart mitochondrial creatine kinase isoenzymes but has antigenic similarity with soluble cytoplasmic brain BB. The situation may be analogous to that found with rat heart mitochondria and rat heart cytoplasmic isoenzymes which we have shown to exhibit antigenic similarity even though differences in electrophoretic and amino acid composition have been demonstrated; however, the confident determination that the particulate enzyme is a separate isoenzyme will have to await amino acid analysis.  相似文献   

5.
The degree to which developmentally related alterations in cardiac creatine kinase (CK) activity reflect modification of CK isoenzyme gene expression remains uncertain. The present studies addressed this question by assessing multiple aspects of CK in rat heart during the perinatal to adult transition. In addition to whole tissue, isolated and purified muscle and nonmuscle cells were studied, as well as myofibrillar, mitochondrial, and cytosolic subcellular fractions. Whole homogenate CK enzyme specific activity nearly doubled during the weanling to adult developmental period. Muscle cell CK activity increased by a similar magnitude. Nonmuscle cell activity decreased. In the adult heart, both myofibrillar and mitochondrial CK activities were augmented versus the weanling heart. The cytoplasmic fraction activity held constant during development. Electrophoretic isoenzyme analyses of both weanling and adult cardiac muscle cells indicated the presence of mitochondrial CK and MM-CK isoforms. Weanling heart nonmuscle cells contained mitochondrial, MM, MB, and BB isoforms; however, BB isoform was not detected in the adult heart nonmuscle cells. Arrhenius plots provided information regarding heart muscle and nonmuscle cell alterations during development. CK activation energies were also determined for whole tissue, muscle/nonmuscle cells, myofibrils, mitochondria, and cytosol. Results demonstrate that heterogeneous muscle/nonmuscle cellular composition and differential myofibrillar/mitochondrial subcellular composition account for normal, developmentally related changes in heart CK enzyme activity. CK isoenzyme gene expression changes were not detected in cardiac muscle cells, and transition of CK-B to CK-M gene expression is limited to nonmuscle cells during normal, weanling to adult development in the rat heart.  相似文献   

6.
Serum and organ creatine phosphokinase alterations in exercise.   总被引:1,自引:0,他引:1  
Rats that swam for 3 h showed a 6-fold increase in serum creatine phosphokinase (SCPK) activity which declined to control values within 7 h after swimming. Of the excess SCPK, 77% was BB isoenzyme; the remainder was mainly MM with traces of MB. Kidney, liver, brain and lung contain mainly BB (50-80%) and only a trace of MB (0-7%). Heart CPK was composed of little BB (8%) and more MB (28%) and MM (64%). Skeletal muscle CPK was almost entirely MM. CPK activity is highest in skeletal muscle, intermediate in heart and brain and lowest in kidney, liver and lung. It is suggested that skeletal muscle and heart are not involved in CPK release in swimming, and kidney, liver and brain may be sites of release.  相似文献   

7.
To examine the role of changes in the distribution of the creatine kinase (CK) isoenzymes [BB, MB, MM, and mitochondrial CK (mito-CK)] on the creatine kinase reaction velocity in the intact heart, we measured the creatine kinase reaction velocity and substrate concentrations in hearts from neonatal rabbits at different stages of development. Between 3 and 18 days postpartum, total creatine kinase activity did not change, but the isoenzyme distribution and total creatine content changed. Hearts containing 0, 4, or 9% mito-CK activity were studied at three levels of cardiac performance: KCl arrest and Langendorff and isovolumic beating. The creatine kinase reaction velocity in the direction of MgATP production was measured with 31P magnetization transfer under steady-state conditions. Substrate concentrations were measured with 31P NMR (ATP and creatine phosphate) and conventional biochemical analysis (creatine) or estimated (ADP) by assuming creatine kinase equilibrium. The rate of ATP synthesis by oxidative phosphorylation was estimated with oxygen consumption measurements. These results define three relationships. First, the creatine kinase reaction velocity increased as mito-CK activity increased, suggesting that isoenzyme localization can alter reaction velocity. Second, the reaction velocity increased as the rate of ATP synthesis increased. Third, as predicted by the rate equation, reaction velocity increased with the 3-fold increase in creatine and creatine phosphate contents that occurred during development.  相似文献   

8.
Primary cultures of newborn rat heart cells were grown for up to 3 weeks in serum-free medium supplemented by insulin, hydrocortisone, transferrin and fetuin. The cells resumed spontaneous beating at 20 h post plating. Mean rates of beating on the second and third day were 79.5 and 94 beats per min, respectively. Cell proliferation occurred during the first 3 days of culture with maximal rates of DNA and protein synthesis on the second day. The highest values of creatine kinase activity were observed on days 2–5 and the three cytoplasmic isozymes, MM, MB and BB, were present in the cultures in proportions similar to those of the newborn heart, indicating stability of the differentiated state of the cells. The relative amount of each isozyme remained unchanged throughout the experiments, MM constituted 70–90% of enzyme activity, MB contributed up to 30% and BB did not exceed 15% of activity. The very low proportion of BB and the lack of increase in this isozyme with age of culture support our earlier morphological observations that non-myocytes do not overgrow the culture.  相似文献   

9.
The interaction of human creatine phosphokinase isoenzymes with rabbit antibodies and their Fab has been studied. It has been shown that Fab of the antibodies against MM or BB isoenzymes preserve high specificity of intact antibodies and the ability to inhibit creatine kinase isoenzymes. Differences between antibodies and their Fab have been found to exist with respect to the kinetics of binding with homologous isoenzymes: the rate of the complex formation for Fab is significantly higher. The interaction of creatine kinase isoenzymes with intact antibodies and their Fab is not affected by the addition of creatine kinase substrates. The antibodies against MM and BB isoenzymes have been used to study the individual properties of each subunit of the M- and B-type in a hybrid dimer MB. It has been shown that such properties of these subunits as the Michaelis constants, pH dependence and inhibition by homologous antibodies are identical to those of non-hybrid MM and BB isoenzymes, respectively.  相似文献   

10.
Properties of human creatine kinase isoenzymes (MM, MB and BB) are investigated. The most pronounced differences in properties of these isoenzymes are found under their urea inactivation, heat denaturation and the inhibition by rabbit antisera to isoenzymes. Differences in values of the Mikhaelis constant and substrate and pH dependencies are much less pronounced. The presence of ADP stabilizes creatine kinase isoenzymes under conditions of urea and heat inactivation. Properties of hybrid MB isoenzymes are found to be intermediate with respect to MM and BB isoenzymes. A mode of the interaction of M and B subunits in dimeric molecules of creatine kinase isoenzymes is discussed.  相似文献   

11.
Abstract: A bioluminescent assay based on the firefly luciferase reaction has been used for determination of creatine kinase activity in CSF. Activities as low as 0.1 U/L can be measured. The coefficient of variation at an activity level of 0.3–0.4 U/L was between 5 and 6%. The assay conditions optimized for serum specimens can be used for CSF. The adenylate kinase activity is almost completely inhibited, which simplifies the procedure. The creatine kinase (CK) isoenzyme distribution was obtained using the bioluminescent assay in combination with immunoinhibition or ion exchange chromatography. All specimens contained both MM and BB activity, but no MB was found. The study indicates that the bioluminescent assay is useful in the determination of CK isoenzymes in CSF. The clinical importance of the observed CK levels will be reported in a separate communication.  相似文献   

12.
Immunochemical and biochemical methods were used to assess quantitatively the changes in the heart creatine kinase system in the myopathic Syrian hamsters, line CHF I46. Cardiomyopathy in I75-200 day old animals was characterized by decreased content of mitochondria and lower total creatine kinase activity. In isolated mitochondria only the creatine kinase activity was decreased while cytochromes aa3 content and respiration rate were unchanged. The share of mitochondrial creatine kinase in the total tissue enzyme activity was decreased from 33% to I8% and that of BB form was elevated from 5% in control to 20%, at unchanged relative level of MM. Immunoassay showed decreased amount of the mitochondrial creatine kinase in the tissue and its decreased ratio to cytochromes aa3. The results show altered expression of creatine kinase isoenzymes in cardiomyopathy.  相似文献   

13.
Creatine kinase isoenzymes from Torpedo californica electric organ, skeletal muscle, and brain were purified and characterized. Torpedo electric organ and skeletal muscle creatine kinase have identical apparent Mr, electrophoretic mobility, and cyanogen bromide fragments. The electrophoretic mobility of the Torpedo creatine kinase was anodal as compared to mammalian MM creatine kinase. No creatine kinase isoenzyme with an electrophoretic mobility similar to mammalian BB creatine kinase was seen in any of the Torpedo tissues examined. Hybridization studies demonstrate the Torpedo electric organ creatine kinase to be composed of identical subunits and capable of producing an enzymatically active heterodimer when combined with canine BB creatine kinase. Creatine kinase from sucrose gradient-purified Torpedo electric organ acetylcholine receptor-rich membranes has an electrophoretic mobility identical with the cytoplasmic isoenzyme and an apparent Mr identical with mammalian MM creatine kinase. Western blot analysis showed Torpedo electric organ skeletal muscle creatine kinase and acetylcholine receptor-enriched membrane creatine kinase reacted with antiserum specific for canine MM creatine kinase. NH2-terminal amino acid sequence determinations show considerable sequence homology between human MM, Torpedo electric organ, chicken MM, and porcine MM creatine kinase. The acetylcholine receptor-associated creatine kinase is, therefore, identical with the cytoplasmic form from the electric organ and is composed of M-subunits.  相似文献   

14.
Cytosolic creatine kinase isoenzymes MM, MB, and BB are assembled from M or B subunits which occur in different relative amounts in specific tissues. The accumulation of mRNAs encoding the M and B subunits was measured during myogenesis in culture. The relative concentration of the two mRNAs was determined by hybridization with a M-CK cDNA probe isolated previously and a B-CK cDNA probe, the cloning and characterization of which is reported here. The B-CK cDNA hybridizes specifically to a 1.6-kb mRNA found in brain and gizzard but not in adult skeletal muscle tissue. The M-CK cDNA hybridizes to a smaller mRNA 1.4-kb long which is specific to skeletal muscle. In culture, the B-CK mRNA is transiently induced and then declines to a low but detectable level.  相似文献   

15.
Mitochondrial creatine kinase was purified from canine myocardium. The preparation exhibited a positively charged isoenzyme free of other creatine kinase isoenzymes and on sodium dodecyl sulfate gel exhibited a single protein band. Amino acid composition showed mitochondrial creatine kinase to be different from that of MM or BB creatine kinase and did not hybridize with the M or B subunits of the cytosolic forms. Antiserum was developed to mitochondrial creatine kinase which did not cross-react with cytosolic creatine kinases. Antiserum to cytosolic creatine kinase exhibited no reaction to mitochondrial creatine kinase. Utilizing the specific antiserum, a radioimmunoassay was developed for the specific detection of mitochondrial creatine kinase. Thus, mitochondrial creatine kinase was purified and shown to be comprised of a unique subunit which is biochemically and immunologically distinct from the cytosolic creatine kinases.  相似文献   

16.
Differentiation of embryonic chick muscle and cultured myogenic cells was studied by the quantitative evaluation of the transition from the embryonic form BB-creatine kinase (CK) to the muscle-specific form MM of CK. Immunoadsorption chromatography was used to establish a method for the quantification of the three isoenzymes MM-CK, MB-CK, and BB-CK in extracts containing all three isoenzymes. The immunoadsorbents were shown to be highly specific for homomeric enzymes; either MM or BB could be prepared in pure form by elution of bound CK from the appropriate adsorbent. The early events in the isoenzyme transition in embryonic breast muscle and myogenic cell cultures were found to be similar. At hatching, however, embryonic muscle contains mainly MM-CK and only traces of MB-CK and BB-CK, whereas cells cultured for 11 days still display a substantial amount of MB-CK and BB-CK.  相似文献   

17.
Kinetic characterization of human heart and skeletal muscle CK isoenzymes   总被引:1,自引:0,他引:1  
C Schneider  G A Stull  F S Apple 《Enzyme》1988,39(4):220-226
The purpose of this study was to investigate the kinetic properties of human creatine kinase (CK) isoenzymes partially purified from heart and skeletal muscle. Utilizing the backward CK-catalyzed reaction of creatine phosphate + ADP in equilibrium creatine + ATP, Km values for heart and skeletal muscle CK MM (3.7 mmol/l) were significantly (p less than 0.05) greater than CK MB (2.1 mmol/l) which were significantly (p less than 0.05) greater than mitochondrial CK (1.8 mmol/l) at variable creatine phosphate and fixed ADP concentrations. However, Km values for similar isoenzymes from the two different tissues, i.e., CK MB from heart vs. skeletal muscle, were not different. These results show that kinetic analysis of CK isoenzymes cannot differentiate the tissue source of elevated blood CK isoenzymes after the acute stress of long distance running or after acute myocardial infarction.  相似文献   

18.
A study of the diagnostic value of serum creatine kinase (CK) isoenzymes showed that MB isoenzyme, which characterizes heart tissue, was a specific and sensitive indicator of acute myocardial infarction. In cases where the clinical picture was complicated by ventricular tachycardia, severe congestive failure, shock, or resuscitation procedures heart, liver, and muscle enzymes were increased. There was also an increase in lactate dehydrogenase isoenzyme values in these cases; indeed, the only enzyme test that correlated well with electrocardiographic and necropsy findings was the MB isoenzyme.  相似文献   

19.
J A Bittl  J DeLayre  J S Ingwall 《Biochemistry》1987,26(19):6083-6090
Brain, heart, and skeletal muscle contain four different creatine kinase isozymes and various concentrations of substrates for the creatine kinase reaction. To identify if the velocity of the creatine kinase reaction under cellular conditions is regulated by enzyme activity and substrate concentrations as predicted by the rate equation, we used 31P NMR and spectrophotometric techniques to measure reaction velocity, enzyme content, isozyme distribution, and concentrations of substrates in brain, heart, and skeletal muscle of living rat under basal or resting conditions. The total tissue activity of creatine kinase in the direction of MgATP synthesis provided an estimate for Vmax (23.4 +/- 2.8, 62.4 +/- 4.5, and 224 +/- 16 mM/s) and exceeded the NMR-determined in vivo reaction velocities by an order of magnitude (4.1 +/- 1.2, 5.1 +/- 1.6, and 18.4 +/- 2.4 mM/s for brain, heart, and skeletal muscle, respectively). The isozyme composition varied among the three tissues: greater than 99% BB for brain; 14% MB, 61% MM, and 25% mitochondrial for heart; and 98% MM and 2% mitochondrial for skeletal muscle. The NMR-determined reaction velocities agreed with predicted values from the creatine kinase rate equation (r2 = 0.98; p less than 0.001). The concentrations of free creatine and cytosolic MgADP, being less than or equal to the dissociation constants for each isozyme, were dominant terms in the creatine kinase rate equation for predicting the in vivo reaction velocity. Thus, we observed that the velocity of the creatine kinase reaction is regulated by total tissue enzyme activity and by the concentrations of creatine and MgADP in a manner that is independent of isozyme distribution.  相似文献   

20.
The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.  相似文献   

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