Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.4.1 (myosin ATPase)
1,140 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Muscle biopsies of the vastus lateralis muscle taken before and after 18 weeks of resistance training were compared by preparing frozen cross sections for electron microscopy and using adjacent sections for fiber typing by myosin ATPase activity. Quantitative ultrastructural changes were observed in histochemically-identified muscle fiber types of twelve young women who underwent the training. The percentage of type IIB fibers decreased and IIA fibers increased. The cross-sectional area of all major fiber types increased with training. The absolute volume of myofibrils, intermyofibrillar space, and mitochondria increased with training for most major fiber types (type I, IIA and IIAB), but the relative volume percentages were not significantly changed because of corresponding fiber hypertrophy. Mean mitochondrial size for types I and IIA and myofibril size for types IIC and IIB increased significantly with training. The capillary number per fiber and density did not change with training. Activity levels were measured for selected glycolytic and oxidative enzymes. Cytochrome oxidase and hexokinase increased significantly with training, while creatine kinase, citrate synthase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase and hydroxyacyl CoA dehydrogenase enzymes were not significantly altered. The results suggest that this type of high-repetition resistance training causes the intracellular components of all fiber types to increase proportionally with an increase in fiber size. In addition, the enzyme analysis indicates the muscle as a whole may increase its oxidative phosphorylation capacity in conjunction with the decreased percentage of type IIB fibers.
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PMID:Muscle fiber types of women after resistance training--quantitative ultrastructure and enzyme activity. 825 33

To clarify the changes in creatine kinase M localization along with the progress of myocardial ischemia, immunoelectron microscopic studies were performed using rabbit anti-canine creatine kinase M Fab'-horseradish peroxidase conjugate in 21 dogs. Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery for 15 (n = 5), 30 (n = 5), 60 (n = 5), or 180 (n = 4) minutes. Two dogs were used as normal controls. As we have already demonstrated, most creatine kinase M in normal myocardial cells was localized over the entire A band in association with the thick filament, suggesting that creatine kinase in this region (A-band creatine kinase) was the enzyme coupled with myosin ATPase. After 15 minutes of ischemia, creatine kinase M showed only minimal changes in its location, indicating that A-band creatine kinase still has the ability to couple with myosin ATPase (reversible injury). However, after 30 minutes of ischemia, A-band creatine kinase diffused markedly to the I band (transitional phase), and after 60 minutes of ischemia, it leaked out to extracellular spaces (irreversible injury). After 180 minutes of ischemia, most A-band creatine kinase disappeared from the myocardial cells (coagulation necrosis). These features of creatine kinase M localization seemed to reflect each stage of ischemic cell injury. We conclude that myocardial ischemia results in a dissociation of creatine kinase molecules from the thick filament, which leads the energy transport system to destruction.
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PMID:Changes in creatine kinase M localization in acute ischemic myocardial cells. Immunoelectron microscopic studies. 840 63

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.
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PMID:Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. 863 32

Recently, the authors have shown that marked necrosis and fibrosis of myocardium were observed in rats given alkaline ionized water (AKW). To clarify the cause of myocardial lesions, the activities of myosin ATPase, actomyosin ATPase and creatine kinase (CK) in myocardium of rats given AKW at 15 weeks-old were compared with those in myocardium of rats given tap water (TPW). Furthermore, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of myocardiac myosin and isoelectric focusing (IEF) of myocardiac CK were performed which revealed a distinct difference between AKW and TPW groups. The activities of myosin ATPase and actomyosin ATPase in the AKW group were higher than those in the TPW group, and these elevated activities were caused by the degradation of myosin in the AKW group judging from the SDS-PAGE pattern of myosin. On the other hand, the activity of CK in the AKW group was lower than that in the TPW group, and the IEF pattern of CK showed leakage of myocardiac CK. These results indicate that increases in actomyosin ATPase activity and myosin ATPase activity, plus the decrease in CK activity caused the disorder of coupled reaction in male rats given AKW at 15 weeks-old. It is concluded that this disorder of coupled reaction may cause marked myocardiac necrosis and fibrosis in rats given AKW.
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PMID:Degradation of myocardiac myosin and creatine kinase in rats given alkaline ionized water. 952 51

Creatine kinase (CK) isoenzymes are present in all vertebrates. An important property of the creatine kinase system is that its total activity, its isoform distribution, and the concentration of guanidino substrates are highly variable among species and tissues. In the highly organized structure of adult muscles, it has been shown that specific CK isoenzymes are bound to intracellular compartments, and are functionally coupled to enzymes and transport systems involved in energy production and utilization. It is however, not established whether functional coupling and intracellular compartmentation are present in all vertebrates. Furthermore, these characteristics seem to be different among different muscle types within a given species. This study will review some of these aspects. It has been observed that: (1) In heart ventricle, CK compartmentation and coupling characterize adult mammalian cells. It is almost absent in frogs, and is weakly present in birds. (2) Efficient coupling of MM-CK to myosin ATPase is seen in adult mammalian striated muscles but not in frog and bird heart where B-CK is expressed instead of M-CK. Thus, the functional efficacy of bound MM-CK to regulate adenine nucleotide turnover within the myofibrillar compartment seems to be specific for muscles expressing M-CK as an integral part of the sarcomere. (3) Mi-CK expression and/or functional coupling are highly tissue and species specific; moreover, they are subject to short term and long term adaptations, and are present late in development. The mitochondrial form of CK (mi-CK) can function in two modes depending on the tissue: (i) in an <<ADP regeneration mode>> and (ii) in an <<ADP amplification mode>>. The mode of action of mi-CK seems to be related to its precise localization within the mitochondrial intermembrane space, whereas its amount might control the quantitative aspects of the coupling. Mi-CK is highly plastic, making it a strong candidate for fine regulation of excitation-contraction coupling in muscles and for energy transfer in cells with large and fluctuating energy demands in general. (4) Although CK isoforms show a binding specificity, the presence of a given isoform within a tissue or a species only, does not predict its functional role. For example, M-CK is expressed before it is functionally compartmentalized within myofibrils during development. Similarly, the presence of ubiquitous or sarcomeric mi-CK isoforms, is not an index of functional coupling of mi-CK to oxidative phosphorylation. (5) Amongst species or muscles, it appears that a large buffering action of the CK system is associated with rapid contraction and high glycolytic activity. On the other hand, an oxidative metabolism is associated with isoform diversity, increased compartmentation, a subsequent low buffering action and efficient phosphotransfer between mitochondria and energy utilization sites. It can be concluded that, in addition to a high variation of total activity and isoform expression, the role of the CK system also critically depends on its intracellular organization and interaction with energy producing and utilizing pathways. This compartmentation will determine the high cellular efficiency and fine specialization of highly organized and differentiated muscle cells.
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PMID:Functional coupling of creatine kinases in muscles: species and tissue specificity. 974 24

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters phi and psi that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible 'coupling' are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when phi or psi is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when psi or psi is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless phi = psi = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the 'adenine nucleotide flux' at the ends of the model into 'creatine flux' in the middle; the overall net flux is small, but only zero if phi = psi. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.
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PMID:Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/myofibril system in muscle. 974 25

The aim of this study was to evaluate myofibrillar creatine kinase (EC 2.7.3.2) activity on the background of the effect of substrate channeling by myosin ATPase and to compare it with creatine kinase (CK) activity of whole skinned fibers. In order to assess CK activity, skinned fibers were prepared from the rat psoas major muscles defined by light microscopy. The activity in permeabilized fibers after treatment with saponin, Triton X-100 and Ca(2+)-free medium reached 2.80, 6.97 and 3.32 micromol ATP min(-1) mg(-1) protein, respectively, when a coupled enzyme assay system with external hexokinase and glucose-6-phosphate dehydrogenase was used. Transmission electron microscopy (TEM) revealed a possible interference among activities of sarcolemmal, sarcoplasmic, myofibrillar and mitochondrial CK from persisting structures. For evaluation of the myofibrillar CK itself, a pure myofibrillar fraction was prepared. Fraction purity was confirmed by TEM and by enzymatic assays for marker enzymes. Two procedures, i.e. the coupled enzyme assay and the evaluation of phosphocreatine (PCr) concentration before and after the CK reaction, were used for measurement of CK activity in this fraction. The procedures resulted in 3.2 nmol ATP min(-1) mg(-1) protein and 7.6 nmol PCr min(-1) mg(-1) protein, respectively. These alternative approaches revealed a discrepancy between the reacting portions of PCr by more than 50 %, which provides information about the size of the effect, generally described as substrate channeling.
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PMID:Creatine kinase reaction in skinned rat psoas muscle fibers and their myofibrils. 1047 Aug 63

Several works have shown the importance of the creatine kinase (CK) system for cardiac energetics and Ca2+ homeostasis. Nevertheless, CK-deficient mice have cardiac function close to normal, at least under conditions of low or moderate workload. To characterize possible adaptive changes of the sarcoplasmic reticulum (SR) and potential role of glycolytic support in cardiac contractility we used the skinned fibre technique to study properties of the SR and myofibrils, in control and muscle-type homodimer (MM-/mitochondrial-CK)-deficient mice. In control fibres, SR Ca2+ loading with ATP and phosphocreatine (solution PL) was significantly better than loading with ATP alone (solution AL), as determined by analysis of caffeine-induced tension transients. Loading in the presence of ATP and glycolytic intermediates (solution GL) was not significantly different from solution PL. These data indicate that Ca2+ uptake by the SR in situ depends on a local ATP:ADP ratio that is controlled by both CK and glycolytic enzymes. In CK-deficient mice, Ca2+ loading was impaired in solution PL due to the absence of CK. In solution GL, loading was significantly increased, such that calculated Ca2+ release parameters were normalized to those in control fibres in solution PL. In CK-deficient mice, fibre kinetic parameters of tension recovery were impaired after quick stretch in solution PL and were not improved in solution GL. These results show that in CK-deficient mice, at least under basal conditions, glycolysis can replace the CK system in fueling the SR Ca2+ ATPase, but not the myosin ATPase, and may in part explain the limited phenotypic alterations seen in the hearts of these mice.
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PMID:Glycolysis supports calcium uptake by the sarcoplasmic reticulum in skinned ventricular fibres of mice deficient in mitochondrial and cytosolic creatine kinase. 1088 44

Various protocols for the isolation and cultivation of adult rat cardiomyocytes were compared, and the cytoprotective potential of the reversible myosin ATPase inhibitor butanedione monoxime (BDM) was evaluated based on cell yield, cell vitality, lactate dehydrogenase (LDH) and creatine kinase (CK) release, and the mRNA expression of atrial natriuretic peptide (ANP). Overall, a yield of 11.9 x 10(6)cells with >92% cell vitality was obtained when BDM was added to the isolation and cultivation buffers. In contrast, cell vitality ranged from 30% to 70% and cell yield was (4-10) x 10(6) when standard methods for the isolation of cardiomyocytes were used. Butanedione monoxime, at a 15 mM concentration, was cytoprotective during the isolation and cultivation of heart muscle cells, as judged by the morphological appearance (rod shape, lack of bleb formation, and other cytoskeleton defects) and the mRNA expression of the ANP gene. The activities of LDH and CK were also significantly reduced (p < 0.05%) when BDM was added to the isolation and cultivation buffer. The results obtained with BDM warrant further investigation into its cytoprotective potential during ischemia and damage to the cytoskeleton.
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PMID:Butanedione monoxime increases the viability and yield of adult cardiomyocytes in primary cultures. 1221 98

The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 +/- 33 nmol PCr min(-1) mg(-1) at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue.
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PMID:Substrate channelling in a creatine kinase system of rat skeletal muscle under various pH conditions. 1252 49


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