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)

Ischaemic myocardium undergoes calcium-independent contracture at millimolar tissue ATP, though in actomyosin solutions ATP must be reduced to micromolar before rigor complexes form. This contracture is associated with myosin ATPase activity that may contribute to tissue de-energization. Here we used isolated rat cardiomyocytes permeabilized with digitonin to analyse in parallel how rigor and myosin ATPase activity are modulated by metabolic conditions that develop during ischaemia. At pH 7.1 and 37 degrees C rigor and myosin ATPase showed co-ordinated bell-shaped dependence on ATP concentration over 3-1000 microM. Rigor, but not myosin ATPase, was inhibited by acidosis (pH 6.2), indicating reduced efficiency of cross-bridge cycling, while both parameters were stimulated by ADP (< or = 1 mM) and unaffected by inorganic phosphate (Pi, 30 mM), AMP, Mg2+, lactate or inhibition of adenylate kinase with diadenosine pentaphosphate. Combined acidosis and high ADP inhibited rigor, while Pi attenuated the enhancement of rigor by ADP. Thus, rigor complex formation activates myosin ATPase in the intact myofilament array, modulated by ADP, Pi and acidosis in the ranges that occur in ischaemia. There was no evidence that adenylate kinase might attenuate falling ATP/ADP ratio at the myofilaments. In combination these effects are sufficient to resolve the apparent discrepancy between ATP concentrations triggering rigor in actomyosin and onset of contracture in ischaemic myocardium. Since rigor contracture activates myosin ATPase it is likely to exacerbate ATP depletion and thereby limit vital cell functions. This positive feedback is consistent with the abrupt depletion of ATP observed in individual cardiomyocytes undergoing deenergization contracture.
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PMID:Modulation of rigor and myosin ATPase activity in rat cardiomyocytes. 971 Aug 3

Actin binding to skeletal muscle myosin subfragment-1 (S1) increases the dissociation rate of reaction products from the myosin ATPase site; conversely, ATP binding facilitates dissociation of complexed acto-S1. However, details of the molecular mechanism by which the ATP- and actin-binding sites communicate with each other is still obscure. We present evidence that the effect of actin is mediated by a conformational change in the loop containing amino acids from 677 to 689 [loop M (677-689)], a segment of the 20-kDa tryptic fragment that contributes to the structure of the ATP-binding cleft. Initially, a fluorescent ADP analogue, methylanthranyloyl-8-azido-ADP (Mant-8-N3-ADP), was covalently crosslinked to loop M (Mant-S1), perhaps at Lys 681. Actin-activated Mg2+-ATP hydrolysis by Mant-S1 was accelerated approximately 6 times over that by unmodified S1, suggesting that the ATPase site is not blocked by the ADP analogue crosslinked in the loop M (677-689). Nevertheless, analysis of Mant-group fluorescence polarization and acrylamide-induced quenching showed the crosslinked probe to be entrapped within the ATP-binding cleft at a location where Mant-group rotational mobility was hindered, and where it was relatively inaccessible to the solvent. Exposing Mant-S1 to Mg2+-ATP and/or actin elicited similar decreases in fluorescence polarization, indicating increased rotational mobility of the Mant-group and movement of crosslinked Mant-8-N3-ADP to a less hindered position. Stern-Volmer quench curves showed that Mant-8-N3-ADP was translocated to a site where it was more accessible to dissolved quencher, perhaps outside the ATP-binding cleft. Since actin does not bind to the ATPase site, actin-induced translocation of Mant-8-N3-ADP crosslinked to loop M (677-689) probably results from a conformational change in loop M (677-689). These results suggest that loop M acts as a signal transducer mediating communication between the ATP- and actin-binding sites.
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PMID:A unique loop contributing to the structure of the ATP-binding cleft of skeletal muscle myosin communicates with the actin-binding site. 972 61

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

Resonance energy transfer probes were attached to skeletal myosin's nucleotide site and regulatory light chain (RLC) to examine nucleotide analog-induced structural transitions. A novel chemical modification of the RLC was developed for specific labeling of the basic N-terminus without affecting myosin ATPase activity. The modification allows attachment of a terbium chelate to rabbit skeletal RLC and was mapped by tryptic digestion to an amino group on the six N-terminal RLC residues. The use of terbium as a resonance energy transfer donor allowed the determination of the efficiency of energy transfer by sensitized emission lifetime measurements that practically eliminate background from unlabeled donor and acceptor sites as well as potential orientation factor artifacts in the calculation of the critical transfer distance. The nucleotide site was labeled with a functional CY3-labeled nucleotide as an energy transfer acceptor. Of the nucleotide states examined, ADP, ADP. vanadate, ADP. A1F4, and ADP. BeFx, the difference between the ADP and ADP. vanadate states was greatest (0.4-nm change), but was not considered to be statistically significant. The binding of actin to ADP-myosin also failed to produce a statistically significant change (0.3-nm change). These results are not consistent with a number of versions of the swinging lever arm hypothesis.
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PMID:Domain motion between the regulatory light chain and the nucleotide site in skeletal myosin. 984 69

In the presence of MgADP, a novel phosphate analogue of gallium fluoride (GaFn) forms a ternary complex with the myosin subfragment-1 (S-1), in the same way that has been previously reported with aluminum fluoride (AlF4-), beryllium fluoride (BeFn), scandium fluoride (ScFn), and vanadate (Vi), and this complex formation may mimic different states along the ATPase kinetic pathway. This novel complex has been characterized and compared with other complexes to ascertain whether it forms a transition-state analogue of myosin ATPase. The complex formed quickly, although several times slower than the BeFn complex. The half-life of the myosin.ADP.GaFn complex was about 50 h at 4 degreesC. The formation of the myosin.ADP.GaFn complex was accompanied by an increase in tryptophane fluorescence, similar to that observed upon the addition of ATP, but slightly lower than that of the M**.ADP.Pi complex. Upon addition of GaFn to acto-myosin.ADP, acto-myosin did not dissociate, and the S-1.ADP.GaFn complex was scarcely decomposed by actin, like the AlF4- and ScFn complexes but unlike the BeFn and Vi complexes. The conformations at the localized region of SH1, SH2, and RLR, which are very accessible to the binding of ATP, were studied by fluorescent labeling and chemical modification, and the results suggested that these conformations are very similar to that of the M**.ADP.Pi state. Small-angle X-ray solution scattering showed that the radius of gyration value decreases by about 3 A when S-1 forms an S-1.ADP.GaFn complex, suggesting that the shape of the complex becomes compact or rounded in shape, similar to that in the presence of ATP or complexes with other phosphate analogues, and thus mimics the myosin**.ADP.Pi state closely. The overall results may indicate that the complex mimics a somewhat different transient state from that of other complexes but has a similar global conformation along the ATPase kinetic pathway.
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PMID:Formation of the myosin.ADP.gallium fluoride complex and its solution structure by small-angle synchrotron X-ray scattering. 988 Aug 15

This review focuses on experiments in which the single turnover of myosin-bound ADP is used to characterize the regulation of the cross-bridge cycle by myosin light chain phosphorylation in mammalian smooth muscle. Under isometric conditions, at rest, when the myosin light chain is not phosphorylated, myosin cycles very slowly (about 0.004 s-1), while phosphorylation of the light chain results in a 50-fold increase in cycling rate of 0.2 s-1. Experiments consistently show that some myosin does not increase its cycling rate although its light chain is phosphorylated. Studies at low levels of myosin light chain phosphorylation show that phosphorylation also induces an increase in the cycling rate of unphosphorylated myosin. The fast cycling phosphorylated myosin is the main determinant of suprabasal myosin ATPase activity, while the cycling rate of cooperatively activated unphosphorylated myosin is slow and appears to depend on the extent of phosphorylation of the entire thick filament. Single turnover experiments measuring the rate of phosphorylation and dephosphorylation of myosin light chain show that the turnover of light chain phosphate can be very rapid (0.3-0.4 s-1) at suprabasal calcium concentrations. The expected effect of such a rapid turnover of light chain phosphorylation on the turnover of myosin-bound ADP is not observed. The effects of low levels of myosin light chain phosphorylation on the single turnover of myosin suggest that the same small pool of myosin remains phosphorylated for relatively long periods of time rather than the entire pool of myosin spending a small fraction of its cycle time in the phosphorylated state.
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PMID:Control of cross-bridge cycling by myosin light chain phosphorylation in mammalian smooth muscle. 988 63

Magnesium (Mg2+) is the physiological divalent cation stabilizing nucleotide or nucleotide analog in the active site of myosin subfragment 1 (S1). In the presence of fluoride, Mg2+ and MgADP form a complex that traps the active site of S1 and inhibits myosin ATPase. The ATPase inactivation rate of the magnesium trapped S1 is comparable but smaller than the other known gamma-phosphate analogs at 1.2 M-1 s-1 with 1 mM MgCl2. The observed molar ratio of Mg/S1 in this complex of 1.58 suggests that magnesium occupies the gamma-phosphate position in the ATP binding site of S1 (S1-MgADP-MgFx). The stability of S1-MgADP-MgFx at 4 degrees C was studied by EDTA chase experiments but decomposition was not observed. However, removal of excess fluoride causes full recovery of the K+-EDTA ATPase activity indicating that free fluoride is necessary for maintaining a stable trap and suggesting that the magnesium fluoride complex is bonded to the bridging oxygen of beta-phosphate more loosely than the other known phosphate analogs. The structure of S1 in S1-MgADP-MgFx was studied with near ultraviolet circular dichroism, total tryptophan fluorescence, and tryptophan residue 510 quenching measurements. These data suggest that S1-MgADP-MgFx resembles the M**.ADP.Pi steady-state intermediate of myosin ATPase. Gallium fluoride was found to compete with MgFx for the gamma-phosphate site in S1-MgADP-MgFx. The ionic radius and coordination geometry of magnesium, gallium and other known gamma-phosphate analogs were compared and identified as important in determining which myosin ATPase intermediate the analog mimics.
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PMID:Inhibition of myosin ATPase by metal fluoride complexes. 1008 41

It is known that ternary complexes of myosin subfragment 1 (S1) with ADP and the Pi analogs beryllium fluoride (BeFx) and aluminum fluoride (AlF4-) are stable analogs of the myosin ATPase intermediates M* x ATP and M** x ADP x Pi, respectively. Using kinetic approaches, we compared the rate of formation of the complexes S1 x ADP x BeFx and S1 x ADP x AlF4- in the absence and in the presence of F-actin, as well as of the interaction of these complexes with F-actin. We show that in the absence of F-actin the formation of S1 x ADP x BeFx occurs much faster (3-4 min) than that of S1 x ADP x AlF4- (hours). The formation of these complexes in the presence of F-actin led to dissociation of S1 from F-actin, this process being monitored by a decrease in light scattering. The light scattering decrease of the acto-S1 complex occurred much faster after addition of BeFx (during 1 min) than after addition of AlF4- (more than 20 min). In both cases the light scattering of the acto-S1 complex decreased by 40-50%, but it remained much higher than that of F-actin measured in the absence of S1. The interaction of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes with F-actin was studied by the stopped-flow technique with high time resolution (no more than 0.6 sec after mixing of S1 with F-actin). We found that the binding of S1 x ADP x BeFx or S1 x ADP x AlF4- to F-actin is accompanied by a fast increase in light scattering, but it does not affect the fluorescence of a pyrene label specifically attached to F-actin. We conclude from these data that within this time range a "weak" binding of the S1 x ADP x BeFx and S1 x ADP x AlF4- complexes to F-actin occurs without the subsequent transition of the "weak" binding state to the "strong" binding state. Comparison of the light scattering kinetic curves shows that S1 x ADP x AlF4- binds to F-actin faster than S1 x ADP x BeFx does: the second-order rate constants for the "weak" binding to F-actin are (62.8 +/- 1.8) x 10(6) M-1 x sec-1 in the case of S1 x ADP x AlF4- and (22.6 +/- 0.4) x 10(6) M-1 x sec-1 in the case of S1 x ADP x BeFx. We conclude that the stable ternary complexes S1 x ADP x BeFx and S1 x ADP x AlF4- can be successfully used for kinetic studies of the "weak" binding of the myosin heads to F-actin.
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PMID:Use of stable analogs of myosin ATPase intermediates for kinetic studies of the "weak" binding of myosin heads to F-actin. 1049 2

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.
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PMID:Movement of smooth muscle tropomyosin by myosin heads. 1050 1


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