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.     

Evaluation of energy metabolism on the myocardium analyzing of creatine kinase isoenzymes in rats]

The purpose of this study is to evaluate the efficiency of energy metabolism in the myocardium by means of the activities of creatine kinase (CK) and CK isoenzyme following age-related change in the heart and experimental heart failure. Wistar strain male rats aged from 3 weeks to 57 weeks after birth were used. The main experimental results obtained are as follows. (1) There were differences in the compositions of CK isoenzyme of the ventricular, atrial, and papillar muscles. No apparent variation, however, was noted among the basal portions of the left and right ventricular muscles, free wall of the left ventricle, and apex. (2) Compositions of CK isoenzyme analyzed from the ventricle and atrium were clearly different. The level of CK-B subunit activity of the ventricular muscle was highest level in the 5-week-old rat, and subsequently dropped significantly in the 24-week-old rat. Thereafter, the level gradually increased with aging. Dramatic change in the energy metabolism in the myocardium occurred in rats more than 3 weeks old. (3) Decrease in activity of m-CK but increase in activity of succinate dehydrogenase analyzed from the ventricular muscle of experimental heart failure induced by monocrotaline was recognized. From these results, the author assumed that the trend of the composition of CK isoenzyme is one of the indices in the determination of the regulation of energy metabolism of the myocardium.     

DNA synthesis in the brain and liver of rats in various stages of postnatal development]

The intensity of incorporation of 3H- and 14C-thymidine in the brain and liver DNA in rats of different ages was investigated. It was proved that both the replicative and oxyurea-resistant DNA synthesis might proceed in the rat brain cells. The intensity of these processes changes sharply during postnatal development.     

Specific features of satellite cells and myoblasts at different stages of rat postnatal development

A cell culture consisting mainly of satellite cells and mononuclear myoblasts was derived from femoral muscles of infant (aged 3–7 days) and adult rats. Satellite cells identified by expression of the specific marker Pax7 accounted for approximately 80% of the isolated cell fraction. Mononuclear myoblasts represented by proliferating and postmitotic cell pools were identified immunocytochemically by the expression of markers Ki67 and desmin. Differentiation of satellite cells and myoblasts in the culture depended on the concentration of Ca²⁺ in the culture medium (F12 with different Ca²⁺ concentrations or DMEM). Differentiation of myogenic cells manifested in myoblasts fusion, formation of myotubes, and expression of myosin in myofibrils was observed only in the medium with a high Ca²⁺ concentration (2mM). Satellite cells and myoblasts from the muscles of newborn and adult rats did not differ noticeably in their capacity for differentiation.     

Dissimilarity in the folding of human cytosolic creatine kinase isoenzymes

Creatine kinase (CK, EC 2.7.3.2) plays a key role in the energy homeostasis of excitable cells. The cytosolic human CK isoenzymes exist as homodimers (HMCK and HBCK) or a heterodimer (MBCK) formed by the muscle CK subunit (M) and/or brain CK subunit (B) with highly conserved three-dimensional structures composed of a small N-terminal domain (NTD) and a large C-terminal domain (CTD). The isoforms of CK provide a novel system to investigate the sequence/structural determinants of multimeric/multidomain protein folding. In this research, the role of NTD and CTD as well as the domain interactions in CK folding was investigated by comparing the equilibrium and kinetic folding parameters of HMCK, HBCK, MBCK and two domain-swapped chimeric forms (BnMc and MnBc). Spectroscopic results indicated that the five proteins had distinct structural features depending on the domain organizations. MBCK BnMc had the smallest CD signals and the lowest stability against guanidine chloride-induced denaturation. During the biphasic kinetic refolding, three proteins (HMCK, BnMc and MnBc), which contained either the NTD or CTD of the M subunit and similar microenvironments of the Trp fluorophores, refolded about 10-fold faster than HBCK for both the fast and slow phase. The fast folding of these three proteins led to an accumulation of the aggregation-prone intermediate and slowed down the reactivation rate thereby during the kinetic refolding. Our results suggested that the intra- and inter-subunit domain interactions modified the behavior of kinetic refolding. The alternation of domain interactions based on isoenzymes also provides a valuable strategy to improve the properties of multidomain enzymes in biotechnology.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

Functional aspects of creatine kinase isoenzymes in endothelial cells

To characterize the isoenzyme distribution of creatine kinase (CK) in endothelial cells (ECs) and its functional role during substrate depletion, ECs from aorta (AECs) and microvasculature (MVECs) of pig and rat were studied. In addition, high- energy phosphates were continuously monitored by (31)P NMR spectroscopy in pig AECs attached to microcarrier beads. CK activity per milligram of protein in rat AECs and MVECs (0.08 +/- 0.01 and 0.15 +/- 0.08 U/mg, respectively) was <3% of that of cardiomyocytes (6.46 +/- 1.02 U/mg). Rat and pig AECs and MVECs displayed cytosolic BB-CK, but no MM-CK. Gel electrophoresis of mitochondrial fractions of rat and pig ECs indicated the presence of mitochondrial Mi-CK, mostly in dimeric form. The presence of Mi(a)-CK was demonstrated by indirect immunofluorescence staining using Mi(a)-CK antibodies. When perifused with creatine-supplemented medium, phosphocreatine (PCr) continuously increased with time (1.2 +/- 0.6 nmol x h(-1) x mg x protein(-1)), indicating creatine uptake and CK activity. Glucose withdrawal from the medium induced a rapid decrease in PCr, which was fully reversible on glucose addition, demonstrating temporal buffering of an energy deficit. Because both cytosolic and mitochondrial CK isoforms are present in ECs, the CK system may also contribute to energy transduction ("shuttle hypothesis").     

Methylation of liver cell chromatin proteins at different stages of post-natal development of albino rats]

Methylation of acid proteins and various histone fractions of liver cell chromatin was studied. The intensity of methylation of acid proteins of animals, differing in age, was shown to be 8--16 times higher than that for histone methylation, despite the fact that the rates of 2-14C-methionine incorporation into these two groups of proteins were practically the same. The intensity of methylation of acid proteins and total histones significantly increased during the post-natal development of the animals. Histone methylation largely occurred at the expense of the arginine-rich H3 fraction and the H2 fraction with a moderate level of arginine and lysine. Lysine-rich histone fractions were not subjected to methylation. It is assumed that chromatin proteins methylation regulates conformational properties of the complex and matrix properties of the genome.     

Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart

Creatine kinase (CK) exists as a family of isoenzymes in excitable tissue. We studied isolated perfused hearts from mice lacking genes for either the main muscle isoform of CK (M-CK) or both M-CK and the main mitochondrial isoform (Mt-CK) to determine 1) the biological significance of CK isoenzyme shifts, 2) the necessity of maintaining a high CK reaction rate, and 3) the role of CK isoenzymes in establishing the thermodynamics of ATP hydrolysis. (31)P NMR was used to measure [ATP], [PCr], [P(i)], [ADP], pH, as well as the unidirectional reaction rate of PCr--> [gamma-P]ATP. Developmental changes in the main fetal isoform of CK (BB-CK) were unaffected by loss of other CK isoenzymes. In hearts lacking both M- and Mt-CK, the rate of ATP synthesis from PCr was only 9% of the rate of ATP synthesis from oxidative phosphorylation demonstrating a lack of any high energy phosphate shuttle. We also found that the intrinsic activities of the BB-CK and the MM-CK isoenzymes were equivalent. Finally, combined loss of M- and Mt-CK (but not loss of only M-CK) prevented the amount of free energy released from ATP hydrolysis from increasing when pyruvate was provided as a substrate for oxidative phosphorylation.     

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.     

Dynamic age-related changes in the human physique at stages in postnatal development

In 926 children (7 age groups) combined group differences of the signs have been studied by means of a unified standardized method, using the hand length as a linear measure; the conformity index to definitive size has been applied. For newborns proportional discrepancy to the definitive sizes is specific. They have a relatively long, thick and round body, short extremities, large dimensions of the head and abundant adipose subcutaneous deposits. According to the constitutional peculiarities the newborns resemble the children of the digestive type in mature persons--pycnotic or endomorphic. Beginning from birth, including children of early and first childhood (4-7 years of age), age changes of complexion go in two directions: a) dolichomorphy of complexion is realized; for it decreasing diameters of the chest and dimensions of the neck, chest, abdomen, pelvis (in boys), shoulder, arm, hand, foot and decrease in the subcutaneous adipose tissue are specific; b) longitudinal proportions of body and extremity segments change under influence of biomechanical adaptations to physical loads in connection with adaptation to the vertical position.     

Octamers of mitochondrial creatine kinase isoenzymes differ in stability and membrane binding

Octamer stability and membrane binding of mitochondrial creatine kinase (MtCK) are important for proper functioning of the enzyme and were suggested as targets for regulatory mechanisms. A quantitative analysis of these properties, using fluorescence spectroscopy, gel filtration, and surface plasmon resonance, revealed substantial differences between the two types of MtCK isoenzymes, sarcomeric (sMtCK) and ubiquitous (uMtCK). As compared with human and chicken sMtCK, human uMtCK showed a 23-34 times slower octamer dissociation rate, a reduced reoctamerization rate and a superior octamer stability as deduced from the octamer/dimer ratios at thermodynamic equilibrium. Octamer stability of sMtCK increased with temperature up to 30 degrees C, indicating a substantial contribution of hydrophobic interactions, while it decreased in the case of uMtCK, indicating the presence of additional polar dimer/dimer interactions. These conclusions are consistent with the recently solved x-ray structure of the human uMtCK (Eder, M., Fritz-Wolf, K., Kabsch, W., Wallimann, T., and Schlattner, U. (2000) Proteins 39, 216-225). When binding to 16% cardiolipin membranes, sMtCK showed slightly faster on-rates and higher affinities than uMtCK. However, human uMtCK was able to recruit the highest number of binding sites on the vesicle surface. The observed divergence of ubiquitous and sarcomeric MtCK is discussed with respect to their molecular structures and the possible physiological implications.     

Differentiation of creatine kinase MB and IgA-linked BB isoenzymes on electrophoresis

IgA-linked creatine kinase (CK, EC 2.7.3.2) is a macro CK type 1 isoenzyme that has an identical electrophoretic mobility to CK-MB. Its presence has the potential of causing misdiagnosis of myocardial infarction. Mixing anti-CK-B antiserum with the sample prior to electrophoresis did not unequivocally distinguish between the two isoenzymes. Similarly, anti-human IgG and IgM antibodies were also ineffective. However, the IgA-linked isoenzyme band was removed by anti-human IgA antiserum. While anti-CK-M antibodies did not affect the electrophoretic mobility of IgA-linked CK-BB, the antibody eliminated both the CK-MB and CK-MM bands. Thus, specific anti-IgA and anti-CK-M antibodies may be used to establish the presence of the myocardial isoenzyme.     

Fiber type composition of the plantarflexors of giraffes (Giraffa camelopardalis) at different postnatal stages of development

1. A sample of fibers from deep (close to the bone) and superficial (away from the bone) regions of the plantaris (PLT) and medial (MG) and lateral (LG) gastrocnemius muscles of a neonatal, a 17-day-old and an adult giraffe were typed qualitatively as dark or light based on alkaline preincubation myosin ATPase staining properties and then sized. 2. Each muscle at all ages showed a higher percentage and a larger cross-sectional area (CSA) or light ATPase fibers in the deep than the superficial region. This relationship was qualitatively, although not quantitatively, similar to that reported in hindlimb muscles of other mammals. 3. At all ages, the PLT, the deepest muscle in the synergistic group, had the highest relative total CSA of light ATPase fibers among the muscles sampled. 4. At birth, the PLT had an unusually high percentage of light ATPase fibers in comparison to that found in the same muscle of other mammals. With age, the total CSA of light ATPase fibers increased dramatically in the PLT and decreased slightly in the MG and LG. 5. These data suggest that the PLT, especially the deep portion, may functionally replace the soleus muscle which is absent in the giraffe. In addition, the fiber type results demonstrate that the changes in the fiber type composition of individual muscles observed at different postnatal ages in the giraffe are relatively similar to that reported in smaller mammals, suggesting the existence of similar regulatory mechanisms.     

Glucose uptake by chicken embryo hearts at various stages of development

d-glucose, but not l-glucose, was found to readily enter the cells of 5- to 6-day chick embryo heart. This suggests the operation of a specific transport system for glucose. The rate of glucose uptake was found to decrease as development proceeds from 5 to 15 days of development, but no further decrease was found between 15 and 20 days. Uptake of glucose is a saturable process, from 5–6 days of embryonic life on. The large decrease in glucose uptake between 5 and 10 days of development is found to be associated with a fourfold increase in the apparent K_m of the uptake process. From 10 days of development onward, the apparent K_m remains about 40 mM. The rate of 2-deoxyglucose uptake also decreased from 5 to 15 days of embryonic life with no further decrease from 15 to 20 days. Glucose competitively inhibits the uptake of 2-deoxyglucose with a K_i close to the K_m for glucose uptake. The uptake of 2-deoxyglucose is stimulated by physiological levels of insulin as early as 5–6 days, although the extent to which insulin enhances uptake is not quite as great as at 15 days of development.