Developmental changes in creatine phosphokinase isoenzymes in neonatal mouse hearts.

The distribution of creatine phosphokinase isoenzymes differs in extracts of newborn and adult mouse hearts. Electrophoresis on acetate strips reveals the presence of BB, MB, and MM isoenzymes in the 2 day old neonate heart, with relative activities of 4%, 24% and 72% respectively. Beginning at 6 days of age, a fourth isoenzyme, shown to be associated with mitochondria, is seen moving toward the cathode. With age the distribution changes, with BB disappearing by 18 days. By 25 days the relative proportions of MB, MM and mitochondrial CPK have reached 5%, 86% and 9%, respectively, similar to the levels seen in the adult. The late appearance of the mitochondrial isoenzyme may reflect a difference in the requirement of the developing and adult heart for ATP and phosphocreatine.     

Serum and organ creatine phosphokinase alterations in exercise.

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.     

Expression of creatine kinase isoenzymes in myogenic cell lines.

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.     

Differential heat sensitivity of human creatine kinase isoenzymes.

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.     

Properties of human creatine kinase isoenzymes]

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.     

Differentiation of creatine phosphokinase during myogenesis: quantitative fractionation of isozymes

A technique is described for the quantitative measurement of creatine phosphokinase (CPK) isozymes in extracts of chick muscle. The isozymes are fractionated by stepwise elution with increasing salt concentrations from DEAE-Sephadex minicolumns. Isozyme separation was confirmed by polyacrylamide gel electrophoresis followed by enzyme staining. We used this method to determine changes in CPK isozymes during the course of myogenesis in culture. The total specific activity of CPK increases about 20-fold during myogenesis. Quantative analysis of isozyme changes shows that the muscle-specific form (MM) accounts for virtually all of this increase. Activity of MM-CPK is undetectable in 1-day cultures, increases rapidly after myoblast fusion, and comprises more than 70% of total CPK in mature cultures. In contrast, the specific activity of the brain-specific isozyme (BB) remains constant throughout myogenesis. This was interpreted as indicating that the B subunit is expressed in both mononucleated cells and myotubes. We confirmed this by analyzing CPK isozymes in fibroblast cultures and in myotube-enriched cultures. Elimination of most of the mononucleated cells in the cultures produced an increase in the specific activity of CPK, but had no effect on the isozyme pattern and did not decrease the relative amount of the BB isozyme. Pure fibroblast cultures contained very low CPK activity, predominantly the BB isozyme.     

Interaction of human creatine phosphokinase isoenzymes with rabbit antibodies and their Fab-fragments

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.     

Secretion of beta-hexosaminidase by cultured human skin fibroblasts. Kinetics, effect of temperature, cell density, serum concentration and pH.

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.     

Bioluminescent Assay of Total and Brain-Specific Creatine Kinase Activity in Cerebrospinal Fluid

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.     

Changes in the activity of various enzymes of carbohydrate metabolism in the liver and skeletal muscles of pigs during ontogenesis

Hexokinase, glucokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activity was studied in the liver and musculus quadriceps femoris of 110-day foetuses 1, 2, 3, 30 and 60-day piglets and in adult pigs. The activity of all enzymes in the tissues of newborn piglets is considerably higher than in the tissues of foetuses. The activity of hexokinase in both tissues of piglets increases in the first days after birth and lowers by the one month age. The phosphofructokinase activity in the skeletal muscles and the glucokinase one in the pig liver increase during the postnatal development. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in both tissues of pigs increases after birth and then decreases. Glucose metabolism in the pig liver at all stages of odontogenesis proceeds more intensively by the pentose phosphate pathway, and in the skeletal muscles--by glycolytic one.     

Characterization of a brain particulate bound form of creatine kinase

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.     

Changes in some properties of 3':5'-AMP-dependent protein kinase from rat skeletal muscles during physical exercise

During 6-week training of rats the activity of isoenzymes I and II of soluble 3':5'-AMP-dependent protein kinase increases by 22 and 33%, respectively. A long-term physical load does not cause any significant changes in the activity of both isoenzymes. The maximal activity of the isoenzymes from skeletal muscles of the control and experimental rats is observed at the same concentrations of 3':5'-AMP and pH of 6,0-6,5. During training and under physical load the apparent Km values for ATP of both isoenzymes of 3':5'-AMP-dependent protein kinase do not change significantly, whereas that of V shows an increase. The apparent Km and V values for the histone increase for isoenzyme I obtained from skeletal muscles of trained rats both at rest and under physical load. In case of isoenzyme II the Km value for the histone decreases, while that of V remains unchanged. The changes in the properties of isoenzymes I and II of 3':5'-AMP-dependent protein kinase from skeletal muscles suggest the participation of the enzyme in adaptation to systematic muscular activity.     

Velocity of the creatine kinase reaction in the neonatal rabbit heart: role of mitochondrial creatine kinase

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.     

Identification of a 43-kDa polypeptide associated with acetylcholine receptor-enriched membranes as MM creatine kinase

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.     

Expression of glycolytic isoenzymes in activated human peripheral lymphocytes: cell cycle analysis using flow cytometry

Human peripheral lymphocytes activated with concanavalin A and phorbol myristate ester exhibit an increase in glycolysis on a time-course similar to that for DNA synthesis. Elevated glycolysis is accompanied by increased specific activities of the glycolytic enzymes. Increased enzyme activities are accounted for by the appearance of specific isoenzyme forms (muscle forms) normally expressed in rapidly growing tumor cells or in growth-stimulated cells. In the present study we analyzed the expression of the glycolytic isoenzymes during cell cycle progression of activated human lymphocytes using two-parameter (DNA and protein) flow cytometry. Time-course studies and analysis of subpopulations prepared by elutriation centrifugation showed that the inducible isoenzymes are expressed at low levels or not at all in G0 cells. They are expressed first during the G0 to G1 transition or in early G1. However, expression increases throughout G1, reaching a maximum in S-phase. Thus, induction of glycolytic isoenzymes provides an excellent marker of T-cell activation and progression toward DNA synthesis.     

Effect of denervation on the distribution and developmental transition of phosphoglycerate mutase and creatine phosphokinase isozymes in rat muscles of different fiber-type composition

Phosphoglycerate mutase (PGM) and creatine phosphokinase (CK) occur as three isozymes (types MM, MB and BB) in mammals and these exhibit similar transitions during skeletal muscle development. To study the influence of innervation on this transition and on the maintenance of the isozyme phenotype in mature muscle, we have determined the changes produced by sciatic neurectomy in neonatal and adult rat hindlimb muscles. In 40-day-old rats, denervation decreased both PGM and CK activity, the effect being more pronounced in the fast-twitch extensorum digitorum longus (EDL) and gastrocnemius muscles than in the slow-twitch soleus muscle. It also produced a progressive increase in the proportion of MB- and BB-PGM isozymes in EDL and gastrocnemius but not in soleus, and an increase of MB- and BB-CK isozymes in all three muscles. In 5-day-old rats, denervation prevented the developmental increase of PGM and CK activity in all three muscles. Denervation also prevented the normal decrease in the relative amounts of the MB and BB isozymes of both enzymes which occur during postnatal muscle development. These results can be explained by the different effects of denervation upon slow and fast muscles, and by the distinct distribution of PGM and CK isozymes in rat type I and II muscle fibers.     

Serum creatine kinase isoenzymes in progressive muscular dystrophy

Creatine kinase isoenzymes in sera and muscle biopsies obtained from 50 controls, 72 patients with progressive muscular dystrophy (PMD), 68 patients with other neuromuscular disorders, 17 carriers of Duchenne-type PMD and 15 patients with myocardial infarction were studied. MB isoenzyme was detected in the sera of 58 patients with PMD and 56 out of 61 muscle biopsies. The MB activity varied between 4 and 400 IU/1 or 3.4--22% of total activity. The MB activity was demonstrated in a considerably smaller number of cases with polymyositis, dystrophic myotonia and Kugelberg-Welander disease. The MB isoenzyme in sera of PMD persisted for many years. It is admitted that the MB isoenzyme in the serum of patients with PMD originates chiefly from skeletal muscle.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques. 3. The substrate specificity of isoenzymes of acid phosphatase in m.gastrocnemius of rabbits.

Three distinct isoenzymes of acid phosphatase have been separated from extracts of m.gastrocnemius of normal and of vitamin E deficient rabbits by gel filtration and polyacrylamide gel electrophoresis. These isoenzymes, termed I, II and III, have molecular weights of: 110,000--130,000, 60,000--78,000 and 12,500--14,500. Isoenzymes I and II split the substrates 4-methylumbelliferyl phosphate and naphthol AS-BI phosphate and the activity is strongly increased in the muscles of vitamin E deficient rabbits. Isoenzyme III splits only 4-methylumbelliferyl phosphate and the activity is not increased in the muscles of vitamin E deficient rabbits. The pH-optimum for isoenzymes I and II is 4.8 and for isoenzyme III 5.5. It has been shown that the histochemical semipermeable membrane technique, using substrate naphthol AS-BI phosphate, is a very reliable technique for demonstrating activity of the isoenzymes I and II in tissue sections. On the other hand, activity of isoenzyme III cannot be demonstrated with this histochemical technique. In pathologically altered muscles, the activity of the isoenzymes I and II is greatly increased whilst the activity of isoenzyme III is not significantly altered.     

Interactions of Zn2+ and Mg2+ with rabbit liver fructose 1,6 bisphosphatase.

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.     

Specific proteolytic modification of creatine kinase isoenzymes. Implication of C-terminal involvement in enzymic activity but not in subunit-subunit recognition.

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.