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1.
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
2.
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
3.
Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition. 总被引:4,自引:1,他引:3
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H G Lebherz T Burke J E Shackelford J E Strickler K J Wilson 《The Biochemical journal》1986,233(1):51-56
We are using the isoenzymes of creatine kinase (CK) to investigate the effect of specific proteolytic modification on the abilities of enzyme subunits to establish precise subunit-subunit recognition in vitro. Previous work by others has shown that treatment of the MM isoenzyme of rabbit CK with Proteinase K results in a specific proteolytic modification and inactivation of the enzyme. In the present work, we show that both the MM and BB isoenzymes of chicken CK are also specifically modified by Proteinase K, resulting in over 98% loss of catalytic activity and approx. 10% decreases in subunit molecular masses of the enzymes. Similar reactions appear to occur when the isoenzymes are treated with Pronase E. Limited amino acid sequence analysis of intact and Proteinase K-modified MM-CK suggests that the proteolytic modification results from a single peptide-bond cleavage occurring between alanine residues 328 and 329, about 50 amino acid residues from the C-terminal end; the active-site cysteine residue was recovered in the large protein fragment of modified M-CK subunits. Proteolytically modified M-CK and B-CK subunits were able to refold and reassociate into dimeric structures after treatment with high concentrations of LiCl and at low pH. Thus the proteolytically modified CK subunits retain their ability to refold and to establish precise subunit-subunit recognition in vitro. 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.
V N Malakhov V A Tishchenko I I Efron E A Chukhri? V A Isachenkov 《Biokhimii?a (Moscow, Russia)》1977,42(7):1221-1231
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
6.
The dimeric rabbit muscle isozyme of creatine kinase (MM) is modified by iodoacetamide to produce the inactive dimer (M'M') and then hybridized with native dimeric brain isozyme (BB). The hybrid enzyme (M'B), as isolated by PAGE, has the same Km for both ATP and creatine but half the specific activity of the brain isozyme (BB). Likewise, the hybrid of the modified brain with the native muscle isozyme (MB') has half the activity of the native muscle enzyme. The M'B, MB' and MB hybrid dimers all have essentially the same electrophoretic properties, and their intrinsic fluorescence and CD spectra in the far-ultraviolet region are very similar to those of the homodimers MM and BB. Similar results were obtained for the hybrid (M"B) containing the muscle enzyme subunit modified at both the thiol group with iodoacetamide and the Trp residue with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide and the native brain enzyme submit. The above results suggest strongly the independent catalytic function of the subunit of creatine kinase. 相似文献
7.
Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid 总被引:1,自引:0,他引:1
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
8.
Z A Khuchua R Ventura-Clapier A V Kuznetsov M N Grishin V A Saks 《Biochemical and biophysical research communications》1989,165(2):748-757
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
9.
Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase 总被引:2,自引:0,他引:2
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
10.
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
11.
Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase 总被引:1,自引:0,他引:1
M B Perryman J D Knell J Ifegwu R Roberts 《The Journal of biological chemistry》1985,260(16):9399-9404
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
12.
K von Figura 《Experimental cell research》1978,111(1):15-21
The temporal relation between the fusion of mononucleated myoblasts into multinucleated fibres and the quantitative changes in the activity of creatine kinase isoenzymes was determined in rat skeletal muscle cell cultures. The effect of actinomycin D on the isoenzyme transition was investigated. The activity of creatine kinase in cultures prior to the onset of cell fusion is predominantly of the BB type. During the phase of cell fusion, there is a manyfold increase in creatine kinase activity. This is due to the appearance or great increase in the activity of the MM isoenzyme. During this period, increase in the BB isoenzyme activity is very small. Inhibition of RNA synthesis by actinomycin D shortly before the onset of cell fusion did not prevent cell fusion and isoenzyme transition during the first 6 h following application of the drug. 相似文献
13.
《Small Ruminant Research》2000,35(3):249-253
Electrophoretic separation of creatine kinase (ATP: creatine phosphotransferase, E.C. 2.7.3.2) isoenzymes in agarose gel was made in the serum of 93 sheep of three Slovenian domestic breeds (Solcavska, Pramenka and Bovska breeds). 44 sheep were pregnant between 112 and 135 days at the time of the blood collection. The median value of serum total CK activity for all animals investigated was 82 U/l. After direct immunoinhibition of CK with anti-M-CK monoclonal antibodies the total CK activity remained the same (88 U/l, P = 0.354). There were significant differences among breeds in CK activity for the Solcavska (101 U/l), Bovska (89 U/l) and Pramenka breeds (73 U/l), respectively (Kruskal–Wallis one way analysis of variance, P < 0.01), and between pregnant (105 U/l) and non-pregnant animals (76 U/l), irrespective of the breed (Mann–Whitney rank sum test, P < 0.05). According to electrophoresis, all non-pregnant sheep had exclusively free CK-BB serum bands activity. In all pregnant sheep coupled dimeric BB variant appeared as macro-CK type 1 in the range between 80% and 100% of total CK activity. The present study confirms the existence of an elimination mechanism for CK from the plasma abundance free CK-BB enzyme. 相似文献
14.
L S Nersesova S A Kosian Zh I Akopian 《Zhurnal evoliutsionno? biokhimii i fiziologii》1986,22(1):89-92
Studies have been made of the activity and isoenzymic spectrum of creatine kinase from human placenta at various stages of its development. Pure preparation of the enzyme was obtained which exhibited low specific activity and intermediate (between MB and BB isoenzymes of creatine kinase) electrophoretic mobility. Some of the properties of this enzyme are described and compared to those of creatine kinase from the brain of rabbits. 相似文献
15.
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
16.
The Creatine kinase (CK) SYSTEM represents key in a power exchange mediators the structure capable to plural interactions with the majority energy making (Glycolysis and mitochondriuns) and energy consuming (ATPases) structures at use of one multifunctional metabolits--creatine and providing transport macroergs inside a cell. Mitochondrions CK provides synthesis creatine phosphates (CP) from cytoplasmic creatine and energy mitochondriums ATP. CP energetically also is structurally more favourable than ATphi. The MM, MB and BB isoforms provide splitting Kphi and synthesis ATphi for M-ATPases, Ca-ATPases and Na-K-ATPases accordingly. Questions of regulation of activity of enzyme, both in ontogenesis, and in blood are discussed. 相似文献
17.
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
18.
The use of an immunohistochemical method permits the localization of creatine kinase isoenzymes MM and BB in tissue sections. Frozen sections are first incubated with the specific antiserum and secondly with the soluble antigen under investigation. The antibody fixed creatine kinase can then be visualized by the tetrazolium-salt linked histochemical reaction. In this way CK-BB was found in the smooth muscle and the mucosa of the human colon. In sections of skeletal muscle CK-MM was predominantly localized in the intermyofibrillar space. Membrane bound activity could be demonstrated in the sarcoplasmic reticulum and the surface membrane after elution of the cytoplasmic enzyme. In the human tonsilla CK-BB was localized in lymphatic and epithelial tissues, CK-MM in the muscle fibers. The isoenzyme patterns in single sections of tonsilla were in parallel determined by the immunotitration assay. The results indicate the usefulness of the combined application of histochemistry and immunotitration in serial tissue sections. 相似文献
19.
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
20.
S Pontremoli E Melloni F Salamino B Sparatore B L Horecker 《Archives of biochemistry and biophysics》1978,188(1):90-97
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