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)

Control of mitochondrial respiration depends on ADP availability to the F1-ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP.Pi which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as 'stimulus-response-metabolism' coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca2+ concentrations. The Ca2+ signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca2+ by activation of Ca2+-sensitive dehydrogenases. The Ca2+-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca2+ leads to increased pyridine nucleotide reduction and oxidative phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Control of mitochondrial respiration in muscle. 305 Apr 50

31P-NMR was used to characterise intracellular phosphate pools and their post mortem changes at 7 degrees C in intact red and white cod muscles under anaerobic conditions. A total phosphate content of 55 and 60 mM was observed in red and white muscle, respectively. The concentration of P-creatine was 14 mM in the white and 9 mM in the red muscle, while that of inorganic phosphate, Pi (30 mM), ATP (9 mM), and sugar phosphate (5 mM) were similar in both muscles. During the first 90 min after death, the decrease in P-creatine showed a first order breakdown with a concomitant stoichiometric increase in Pi content, whereas the ATP and sugar phosphate remained the same. The intracellular pH decreased from 7.4 to 7.3 in this period. The steady-state rate constant of myosin ATPase was 0.0054 and 0.0022/min for red and white muscles, respectively. Individuals kept under diminished oxygen tension prior to being killed, showed a reduced P-creatine level in both muscles.
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PMID:31P-NMR studies of phosphate metabolites in intact red and white swimming muscles of cod (Gadus morhua L.). 375 79

The synthesis of [2-3H]ATP with specific activity high enough to use for 3H NMR spectroscopy at micromolar concentrations was accomplished by tritiodehalogenation of 2-Br-ATP. ATP with greater than 80% substitution at the 2-position and negligible tritium levels at other positions had a single 3H NMR peak at 8.20 ppm in 1D spectra obtained at 533 MHz. This result enables the application of tritium NMR spectroscopy to ATP utilizing enzymes. The proteolytic fragment of skeletal muscle myosin, called S1, consists of a heavy chain (95 kDa) and one alkali light chain (16 or 21 kDa) complex that retains myosin ATPase activity. In the presence of Mg2+, S1 converts [2-3H]ATP to [2-3H]ADP and the complex S1.Mg[2-3H]ADP has ADP bound in the active site. At 0 degrees C, 1D 3H NMR spectra of S1.Mg[2-3H]ADP have two broadened peaks shifted 0.55 and 0.90 ppm upfield from the peak due to free [2-3H]ADP. Spectra with good signal-to-noise for 0.10 mM S1.Mg[2-3H]ADP were obtained in 180 min. The magnitude of the chemical shift caused by binding is consistent with the presence of an aromatic side chain being in the active site. Spectra were the same for S1 with either of the alkali light chains present, suggesting that the alkali light chains do not interact differently with the active site. The two broad peaks appear to be due to the two conformations of S1 that have been observed previously by other techniques. Raising the temperature to 20 degrees C causes small changes in the chemical shifts, narrows the peak widths from 150 to 80 Hz, and increases the relative area under the more upfield peak. Addition of orthovanadate (Vi) to produce S1.Mg[2-2H]ADP.Vi shifts both peaks slightly more upfield without changing their widths or relative areas.
J Biomol NMR 1993 May
PMID:[2-3H]ATP synthesis and 3H NMR spectroscopy of enzyme-nucleotide complexes: ADP and ADP.Vi bound to myosin subfragment 1. 835 34

The binding of Ca(2+)- and Ba(2+)-calmodulin to caldesmon and its functional consequence was investigated with three different calmodulin mutants. Two calmodulin mutants have pairs of cysteine residues substituted and oxidized to a disulphide bond in either the N- or C-terminal lobe (C41/75 and C85/112). The third mutant has phenylalanine-92 replaced by alanine (F92A). Binding measurements in the presence of Ca2+ by separation on native gels and by carbodiimide-induced cross-linking showed a lower affinity for caldesmon in all the mutants. When Ca2+ was replaced by Ba2+ the affinity of calmodulin for caldesmon was further reduced. The ability of Ca(2+)-calmodulin to release caldesmon's inhibition of the actin-tropomyosin-activated myosin ATPase was virtually abolished by mutation of phenylalanine-92 to alanine or by replacing Ba2+ for Ca2+ in native calmodulin. Both cysteine mutants retained their functional ability, but the increased concentration needed for 50% release of caldesmon inhibition reflected their decreased affinity. Ca2+ -calmodulin produced a broadening in the signals of the NMR spectrum of the 10 kDa Ca(2+)-calmodulin-binding C-terminal fragment of caldesmon arising from tryptophans -749 and -779 and caused an enhancement of maximum tryptophan fluorescence of 49% and a 16 nm blue shift of the maximum. Ca(2+)-calmodulin F92A produced a change in wavelength of 4 nm but no change in maximum, whereas Ca(2+)-calmodulin C41/75 binding produced a decrease in fluorescence with no shift of the maximum. We conclude that functional binding of Ca(2+)-calmodulin to caldesmon requires multiple interaction sites on both molecules. However, some structural modification in calmodulin does not abolish the caldesmon-related functionality. This suggests that various EF hand proteins can substitute for the calmodulin molecule.
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PMID:Multiple-sited interaction of caldesmon with Ca(2+)-calmodulin. 868 82

The purpose of this study was to examine the relationships between the relative contents of phosphocreatine (PCr), inorganic phosphate (Pi), beta-adenosine triphosphate (ATP), and transverse relaxation time (T2) with fiber composition, which determined histochemically in the human skeletal muscle. The vastus lateralis muscles of 28 volunteers were subjected to phosphorus nuclear magnetic resonance (31P NMR) spectroscopy, magnetic resonance imaging (MRI) and muscle biopsy. Muscle fibers were divided into type I and type II fibers using myosin ATPase stain. A wide range of fiber composition levels were observed in the subjects (27.3-74.6% type I fibers). The PCr/ATP, Pi/ATP and (PCr + Pi)/ATP ratios were positively related to the percentage of type II fibers (r = 0.695, p < 0.001, r = 0.429, p < 0.05 and r = 0.773, p < 0.001, respectively). There was no correlation between fiber composition and the PCr/Pi ratio (r 0.127, n.s.) or intracellular pH (r = 0.305, n.s.). Moreover, no correlation was found between T2 and fiber type (r = 0.144, n.s.). These results suggest that 31P NMR can detect the differences in relative content of phosphates between type I and type II fibers, thereby noninvasively evaluating fiber composition in human skeletal muscle.
NMR Biomed 1996 Feb
PMID:Relationships between fiber composition and NMR measurements in human skeletal muscle. 884 27

We have previously demonstrated in vitro actin movement at nanomolar adenosine triphosphate (ATP) levels using heavy meromyosin from skeletal muscle. In the present work we tested whether the motility at nonomolar ATP-concentrations could be supported by cardiac myosin as well. Actomyosin (skeletal actin and bovine ventricular myosin) was pretreated in the in vitro motility assay with 1 mM ATP; subsequently, the ATP level was reduced by multiple rigor-solution washes. By the final rigor-solution wash, the ATP concentration, monitored by the luciferin-luciferase assay, dropped to the order of 100 nM. Even at this low ATP level actin-filament movement remained in evidence. This was in marked contrast to the situation where ATP concentration was gradually increased from zero; in the latter, filament movement began only as ATP levels exceeded 1-2 microM. The difference indicates that potential energy is stored during the initial ATP treatment, and utilized later as the free ATP falls below micromolar levels. Although the velocity of cardiac myosin-supported movement was only one fourth of that of skeletal myosin, both myosins supported actin movement down to similar ATP concentrations. The similarity in response of the two myosins to ATP implies a similar degree of potential energy storage. Given the significantly different specific ATPase activities, however, it appears that the mechanism of potential energy storage and release involves factors different from those involved in the release of chemical energy by the myosin ATPase.
Physiol Chem Phys Med NMR 1995
PMID:Persisting in vitro actin motility at nanomolar adenosine triphosphate levels: comparison of skeletal and cardiac myosins. 886 77

We have investigated the functional properties of a mutant (Cg1) derived from the C-terminal 99 amino acids of chicken caldesmon, 658-756 (658C) where the sequence 691glu-trp-leu-thr-lys-thr696 is changed to pro-gly-his-tyr-asn-asn. Cg1 bound Ca2+-calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+-calmodulin with the Trp-722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+-calmodulin is not able to reverse the Cg1-induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin-tropomyosin activation of myosin ATPase. The results are interpreted in terms of multisite attachment of actin and Ca2+-calmodulin to overlapping sequences in caldesmon domain 4b.
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PMID:Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction. 950 48

Myosin forms stable ternary complexes with ADP and the phosphate analogues, fluoroaluminate (Al F4-), fluoroberyllate (BeFn) or orthovanadate (Vi); these ternary complexes mimic transient intermediates in the myosin ATPase cycle. Moreover, we previously demonstrated that these complexes may mimic different myosin ATPase reaction intermediates corresponding to separate steps in the cross-bridge cycle [Maruta, S., Henry, G. D., Sykes, B. D. & Ikebe, M. (1993) J. Biol. Chem. 268, 7093-7100]. Park et al. suggested that the changing conformation of ATP during hydrolysis stresses the active site of myosin subfragment-1 (S-1) through protein-nucleotide contacts at the gamma-phosphate and nucleotide base, and the stress-induced strain in the cross-bridge may be the mechanism by which energy in ATP is transferred to the myosin structure [Park, S., Ajtai, K. & Burghardt, T. P. (1997) Biochemistry 36, 3368-3372]. In the present study, the photoactive ADP analogue, 3'-O-(N-methylanthraniloyl)-2-azido-ADP (Mant-2-N3-ADP), and the 19F-labeled ADP analogue, 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate, were employed to examine conformational differences in protein-nucleotide contact in the ATP-binding site that may correlate with energy transduction. Mant-2-N3-ADP was trapped within the active site of skeletal and smooth muscle myosin in the presence of AlF4-, BeFn or Vi. For both skeletal and smooth muscle myosins, trapped Mant-2-N3-ADP was covalently linked to the 25-kDa N-terminal fragment of S-1 of both myosin/Mant-2-N3-ADP/AlF4- and BeFn complexes, presumably at Trp130. However, the efficiency of the incorporation was much higher for skeletal than for smooth muscle myosin suggesting that the conformations of the adenine-binding pockets of the two myosins are somewhat different. Although the amount of Mant-2-N3-ADP trapped in the presence of AlF4- and BeFn was the same for both myosins, the efficiency of photolabeling skeletal muscle myosin was approximately two times higher for BeFn complex than for AlF4- complex. The 19F-NMR spectra of the bound 2-[(trifluoromethylnitrophenyl)aminoethyl]diphosphate in the ternary complexes formed in the presence of AlF4-, BeFn or Vi showed small but distinguishable differences. Taken together, these results indicate that there is some variation in the protein-nucleotide contacts at the nucleotide base among the ternary complexes studied, and these differences mimic separate steps occurring transiently during the contractile cycle.
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PMID:Analysis of stress in the active site of myosin accompanied by conformational changes in transient state intermediate complexes using photoaffinity labeling and 19F-NMR spectroscopy. 954 69

A number of cellular metabolites, including inorganic phosphate and ADP, have been proposed to regulate the contractions of smooth muscle. Hypothesizing that one of these would have a greater influence than the others, parallel experiments using tissue mechanics and (31)P-NMR allowed comparison of several metabolic components with the generation of force in porcine carotid artery smooth muscle during long-term contractions. P(i), ADP, ATP, PCr, free energy, pH, and free Mg(2+) were determined from phosphate spectra during a control-hypoxia-postcontrol sequence generated during K(+) stimulation by replacement of oxygen with nitrogen using either pyruvate or glucose as substrate. Both pH and free Mg(2+) were significantly lower in control pyruvate-supplied tissues than in glucose-supplied tissues. Mechanical experiments following the same protocol produced variations in force. The pyruvate series produced the greater range of mechanical and metabolic changes. Linear and logarithmic regression analysis found the order of correlation with force to be highest for P(i), followed by pH, free energy, PCr, ATP, ADP, and free Mg(2+). The results are consistent with models for the regulation of myosin ATPase by free phosphate inhibition. The results are inconsistent with models of ADP as a regulator of smooth muscle force. Perturbations which alter intracellular phosphate, such as creatine loading, may produce side effects on the contractions of vascular smooth muscle.
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PMID:Influence of cellular energy metabolism on contractions of porcine carotid artery smooth muscle. 1114 7

Recently reported decameric vanadate (V(10)) high affinity binding site in myosin S1, suggests that it can be used as a tool in the muscle contraction regulation. In the present article, it is shown that V(10) species induces myosin S1 cleavage, upon irradiation, at the 23 and 74 kDa sites, the latter being prevented by actin and the former blocked by the presence of ATP. Identical cleavage patterns were found for meta- and decavanadate solutions, indicating that V(10) and tetrameric vanadate (V(4)) have the same binding sites in myosin S1. Concentrations as low as 50 muM decavanadate (5 muM V(10) species) induces 30% of protein cleavage, whereas 500 muM metavanadate is needed to attain the same extent of cleavage. After irradiation, V(10) species is rapidly decomposed, upon protein addition, forming vanadyl (V(4+)) species during the process. It was also observed by NMR line broadening experiments that, V(10) competes with V(4) for the myosin S1 binding sites, having a higher affinity. In addition, V(4) interaction with myosin S1 is highly affected by the products release during ATP hydrolysis in the presence or absence of actin, whereas V(10) appears to be affected at a much lower extent. From these results it is proposed that the binding of vanadate oligomers to myosin S1 at the phosphate loop (23 kDa site) is probably the cause of the actin stimulated myosin ATPase inhibition by the prevention of ATP/ADP exchange, and that this interaction is favoured for higher vanadate anions, such as V(10).
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PMID:Decavanadate as a biochemical tool in the elucidation of muscle contraction regulation. 1552 16


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