Myosin Adenosine Triphosphate Research Articles

F-actin architecture determines the conversion of chemical energy into mechanical work

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Probing actin-activated ATP turnover kinetics of human cardiac myosin II by single molecule fluorescence.

Mechanistic insights into myosin II energy transduction in striated muscle in health and disease would benefit fromfunctional studies of a wide range of point-mutants. This approach is, however, hampered by the slow turnaround of myosin II expression that usually relies on adenoviruses for gene transfer. A recently developed virus-free method is more time effective but would yield too small amounts of myosin for standard biochemical analyses. However, if the fluorescent adenosine triphosphate (ATP) and single molecule (sm) total internal reflection fluorescence microscopy previously used to analyze basal ATP turnover by myosin alone, can be expanded to actin-activated ATP turnover, it would appreciably reduce the required amount of myosin. To that end, we here describe zero-length cross-linking of human cardiac myosin II motor fragments (sub-fragment 1 long [S1L]) to surface-immobilized actin filaments in a configuration with maintained actin-activated ATP turnover. After optimizing the analysis of sm fluorescence events, we show that the amount of myosin produced from C2C12 cells in one 60 mm cell culture plate is sufficient to obtain both the basal myosin ATP turnover rate and the maximum actin-activated rate constant (kcat). Our analysis of many single binding events of fluorescent ATP to many S1L motor fragments revealed processes reflecting basal and actin-activated ATPase, but also a third exponential process consistent with non-specific ATP-binding outside the active site.

A deep learning-based approach for fully automated segmentation and quantitative analysis of muscle fibers in pig skeletal muscle

Muscle fiber properties exert a significant influence on pork quality, with cross-sectional area (CSA) being a crucial parameter closely associated with various meat quality indicators, such as shear force. Effectively identifying and segmenting muscle fibers in a robust manner constitutes a vital initial step in determining CSA. This step is highly intricate and time-consuming, necessitating an accurate and automated analytical approach. One limitation of existing methods is their tendency to perform well on high signal-to-noise ratio images of intact, healthy muscle fibers but their lack of validation on more complex image datasets featuring significant morphological changes, such as the presence of ice crystals. In this study, we undertake the fully automatic segmentation of muscle fiber microscopic images stained with myosin adenosine triphosphate (mATPase) activity using a deep learning architecture known as SOLOv2. Our objective is to efficiently derive accurate measurements of muscle fiber size and distribution. Tests conducted on actual images demonstrate that our method adeptly handles the intricate task of muscle fiber segmentation, yielding quantitative results amenable to statistical analysis and displaying reliability comparable to manual analysis.

Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle

We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247-3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856-6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.

Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30-40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2-3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.

The pre-steady state of the myosin-adenosine triphosphate system. XI. Formation and decomposition of the reactive myosin-phosphate-ADP complex.

The pre-steady state of the myosin-adenosine triphosphate system. XI. Formation and decomposition of the reactive myosin-phosphate-ADP complex.

Adenosine Triphosphate Inhibition in Myosin Adenosine Triphosphate Systems

Abstract Factors affecting inhibition of myosin ATPase by ATP, such as concentration of the enzyme, of ATP, and of the divalent metals, calcium and magnesium, have been studied and the results are reported here. Experiments were carried out under conditions in which no known magnesium effects occurred, so that only the effect of calcium on the nucleoside triphosphatase activity of myosin A could be qualitatively characterized. Myosin A ATPase was inhibited by free ATP at 0.15 ionic strength in the presence of millimolar calcium and 0.006 m KCl. A reaction model is proposed in which Ca-ATP is the substrate for native myosin A and inhibition is brought about by 2 moles of ATP.

The pre-steady state of the myosin-adenosine triphosphate system. IX. Effect of F-actin on the myosin-ATP system.

The pre-steady state of the myosin-adenosine triphosphate system. IX. Effect of F-actin on the myosin-ATP system.

The pre-steady state of the myosin-adenosine triphosphate system. VII. Kinetic studies on the decrease in light-scattering of actomyosin induced by ATP.

The pre-steady state of the myosin-adenosine triphosphate system. VII. Kinetic studies on the decrease in light-scattering of actomyosin induced by ATP.

The pre-steady state of the myosin-adenosine triphosphate system. VI. Effects of ATP concentration, pH and temperature.

After mixing ATP with myosin the reaction was stopped by adding 5 per cent trichloroacetic acid, and the amount of P1 liberated was measured. The change in hydrogen ion concentration was measured by a stopped-flow method at pH 8.3, in which the hydrolysis of ATP by myosin yielded 1 mole of hydrogen ion per mole of ATP hydrolyzed. The following results were obtained from this experiment. 1. At ATP concentrations lower than 1 mole per 4 × 105 g of myosin, both the initial rapid P1- and hydrogen ion-liberations followed mono-molecular kinetics, and their rates were equal. The rates of P1- and hydrogen ion-liberation were independent of the ATP concentration when the concentration was lower than 1 mole per 4 ×105 g of myosin, but they increased with increasing ATP concentration at concentrations above this. 2. The amount of initial rapid P1-liberation increased linearly with the ATP concentration until the amount of added ATP reached about 1 mole per 4 ×105 g of myosin. At higher ATP concentrations, the amount was constant at about 1 mole per 4 ×105g of myosin. 3. At ATP concentrations lower than about 1 mole per 4 ×105 g of myosin, the amount of initial rapid hydrogen ion-liberation also increased with increasing ATP concentration, and reached about 1 mole per 4 × 105 g of myosin at ATP concentrations higher than the stoichiometric amount. 4. The pH-dependence of the rate of initial rapid P1-liberation was different from that of the rate at the steady state: the initial rate increased with increasing pH and the half maximum value was obtained at pH 6.4. 5. The activation entropy of the initial rapid P1- and hydrogen ion-liberations was found to be +21.6 cal per mole per deg., while the activation entropy of P1-liberation at the steady state was −24.8 cal per mole per deg. 6. The rate of the actomyosin type ATPase [EC 3.6. 1.3] reaction increased with increasing pH and the half maximum value was obtained at pH 6.1. The activation entropy of the actomyosin type ATPase reaction was +45.5 cal per mole per deg.

The Pre-steady State of the Myosin-Adenosine Triphosphate System<subtitle>V. Evidence for a Phosphate Exchange Reaction between Adenosine Triphosphate and the “Reactive Myosin-phosphate Complex” <xref ref-type="fn" rid="fn1"><sup>*</sup></xref></subtitle>

In our previous work, it was shown that the initial reaction between myosin-ATPase [EC 3.6.1.3, ATP phosphohydrolase] and ATP produces one mole of “reactive myosin-phosphate complex” (EP) per 4×105g of myosin, which is TCA**-labile. However, there has been found so far no unequivocal evidence for a covalent bonding of phosphate to myosin in the complex. In the present work, therefore, a search was made for a phosphate exchange reaction between ATP and EP. The amount of TCA-labile γ32P1 liberated was measured as a function of time after the reaction was started by adding γ-32 P-labelled ATP to myosin in the ratio of 1 mole to 4×105g (the first, or control, experiment). In a subsequent experiment, 1 mole of non-labelled ATP was added to 4×105 g of myosin, and at an appropriate time thereafter, a small amount of γ-32P-ATP, with about the same radioactivity as that of the ATP-32P used in the control experiment, was added to the myosin-ATP system (the second experiment). The 32P1-liberation was followed after adding TCA to stop the reaction, and was compared with that in the control experiment. There are several lines of evidence either that the Michaelis complex (ES) is in equilibrium with myosin (E) and ATP (S), or that the amount of ES is negligibly small. Therefore, the time-course of TCA-labile 32P1-liberation from the myosin-ATP system in the second experiment can be easily calculated from the time-course of the control experiment, if it is assumed that there is no phosphate-exchange reaction between EP and ATP. The difference between the experimental values and the ones calculated on this assumption can be attributed to the exchange of phosphate between EP and γ-32P-ATP. Myosin was preincubated in 2.8 M KC1 and 10 mM MgCl2 at pH 7.5 and 0°C, prior to the addition of ATP, and it was found that the initial reaction of the myosin-ATP system was not affected by preincubation lasting up to, at least, 3 hr. The time-course of TCA-labile 32P1-liberation in the second experiment was insensitive to a variation in the amount of hot ATP (of constant radioactivity) from 0.005 to 0.05 moles per 4×105 g of myosin, when this hot ATP was added to the system one minute after the addition of 1 mole of cold ATP per 4×105 g of myosin. Therefore, the amount contained in EP of an intermediate exchanging P with the terminal P of ATP could be measured by the method mentioned above. In the presence of 2.8 M KC1 and 10 mM MgCl2 and at p11 7.5 and 0°C, 15–20 percent of the total EP were found to be P-exchangeable with ATP 1 min after the addition of ATP to myosin at a molar ratio of 1: 1. The content of phosphate-exchangeable intermediate in the total EP decreased with decreasing concentrations of KCl and MgCl2: it was 0–10 per cent of EP in the presence of 1.08 M KCI and 5 mM MgCl2, and varied considerably from one preparation to another. It was not affected when the temperature was raised from 0 to 15°C, or when 0.1 mM CaCl2 or 0.1 mM EGTA was added to the reaction mixture. In the presence of 1.1M KCI and 5mM MgCl2 and at pH7.5 and 0°C, the time-course of TCA-labile 32P1-liberation was followed, first, with a system in which 1 mole of hot ATP was added per 4×105 g of myosin, and second, with the systems in which 1 mole of hot ATP per 4×105 g of myosin was added various times after the addition of 1 mole of cold ATP. In this experiment a myosin preparation was used which showed no exchange reaction between EP and ATP under the above conditions. In this case, the initial rate of 32P1-liberation was proportional to the amount of total E minus EP, when corrections were made for the effect of ADP and the rate of ATP-decomposition via simple hydrolysis. Therefore, by analyzing the experimental results described above, the life-time of EP could be easily calculated, and it could be shown that the rate constant of decomposition of EP into myosin and P1 was about 1/5 that of simple hydrolysis of ES.

The Pre-steady State of the Myosin—Adenosine Triphosphate SystemIV. Liberation of ADP from the Myosin—ATP System and Effects of Modifiers on the Phosphorylation of Myosin*

The Pre-steady State of the Myosin—Adenosine Triphosphate System: IV. Liberation of ADP from the Myosin—ATP System and Effects of Modifiers on the Phosphorylation of Myosin Get access KIICHI IMAMURA, KIICHI IMAMURA Search for other works by this author on: Oxford Academic PubMed Google Scholar MICHIHIKO TADA, MICHIHIKO TADA **First Department of Medicine, Osaka University School of MedicineOsaka Search for other works by this author on: Oxford Academic PubMed Google Scholar YUJI TONOMURA YUJI TONOMURA Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 59, Issue 3, March 1966, Pages 280–289, https://doi.org/10.1093/oxfordjournals.jbchem.a128297 Published: 01 March 1966 Article history Received: 17 November 1965 Published: 01 March 1966

The Pre-steady State of the Myosin-Adenosine Triphosphate SystemII. Initial Rapid Absorption and Liberation of Hydrogen Ion Followed by a Stopped-flow Method*

Journal Article The Pre-steady State of the Myosin-Adenosine Triphosphate System: II. Initial Rapid Absorption and Liberation of Hydrogen Ion Followed by a Stopped-flow Method Get access TOMONOBU TOKIWA, TOMONOBU TOKIWA Department of Biology, Faculty of Science, Osaka UnivsrsityOsaka Search for other works by this author on: Oxford Academic PubMed Google Scholar YUJI TONOMURA YUJI TONOMURA Department of Biology, Faculty of Science, Osaka UnivsrsityOsaka Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 57, Issue 5, May 1965, Pages 616–626, https://doi.org/10.1093/oxfordjournals.jbchem.a128124 Published: 01 May 1965 Article history Received: 19 September 1964 Published: 01 May 1965

Molecular kinetics of muscle adenosinetriphosphatase. III. Influence of hydrostatic pressure

1. 1. An experimental study has been made of the kinetics of the myosinadenosine triphosphate system over a range of hydrostatic pressures (up to 10,000 lb./sq. in.). The work was done over a range of substrate concentrations and at three potassium chloride concentrations, namely 0.05, 0.3, and 0.6 M. 2. 2. The data have been analyzed in terms of the Michaelis-Menten mechanism of enzyme action, and volumes of activation calculated for the bimolecular interaction of enzyme and substrate and for the breakdown of the complex into products; these results are summarized in Table II. Some indication has also been obtained of the effect of KCl concentration on the kinetics. 3. 3. The results are shown to be consistent with previous hypotheses as to the nature of the enzyme-substrate interaction and of the breakdown of the complex. The volumes associated with the enzyme-substrate interaction are positive, which is consistent with there being a charge neutralization as the enzyme and substrate come together. The volumes associated with the breakdown are negative, a fact which is attributed to changes in the conformation of the enzyme. 4. 4. The data obtained for this reaction are compared with volume and entropy data for other enzyme reactions, and are found to show some features in common.

Molecular kinetics of muscle adenosine triphosphatase. II. Solvent and structural effects

1. 1. The kinetics of the myosin-adenosine triphosphate system have been studied in various methanol-water and dioxane-water mixtures, at both low and high substrate concentrations. The rates are found to increase as the concentrations of methanol and of dioxane are increased. 2. 2. From the results are calculated the electrostatic contributions to the entropy of formation of the enzyme-substrate complex and to that of the breakdown of the complex. Using the data of a previous paper the corresponding nonelectrostatic entropies are obtained. 3. 3. The results are shown to be consistent with charge formation, while during complex breakdown there is apparently a separation of like (negative) charges.