Evidence for the two-step binding of ATP to myosin subfragment 1 by the rapid-flow-quench method

1. The initial steps on the myosin ATPase (EC 3.6.1.3) pathway are taken to be: (formula; see text) A two-step binding for ATP is assumed, but the evidence for it is unconvincing; because of the rapidity of the process unambiguous values for K1 and K2 are not available. 2. We investigated the myosin mechanism by the chemical flow-quench technique. Reaction mixtures containing [gamma-32P]ATP plus myosin subfragment 1 were quenched in unlabelled ATP (ATP chase) or acid (Pi burst). 3. We show that the ATP-chase method can lead directly to unambiguous values for K1 and k+2. 4. The binding process was slowed down by 40% ethylene glycol. It was studied as a function of the ATP concentration. A limiting plateau resulted, showing a two-step binding for ATP, and values for K1 and k+2 were obtained. 5. K1 and k+2 are rather sensitive to the experimental conditions. Ethylene glycol and lowering of the pH decrease both constants, but an increase in KCl concentration increases them. This suggests that the binding of ATP to myosin is of an electrostatic nature. 6. The Pi-burst method can lead directly to k+3 + k-3, but under certain conditions the kinetics are governed by K1 and k+2. This uncertainty of the interpretation of Pi-burst experiments is discussed.

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Transient kinetics of ATP hydrolysis by covalently crosslinked actomyosin complex in water and 40% ethylene glycol by the rapid flow quench method

Biochemistry ◽

10.1021/bi00335a059 ◽

1985 ◽

Vol 24 (14) ◽

pp. 3814-3820 ◽

Cited By ~ 20

Author(s):

J. A. Biosca ◽

F. Travers ◽

T. E. Barman ◽

R. Bertrand ◽

E. Audemard ◽

...

Keyword(s):

Ethylene Glycol ◽

Atp Hydrolysis ◽

Transient Kinetics ◽

Actomyosin Complex ◽

Rapid Flow ◽

Kinetics Of ◽

Quench Method

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Modification of the interactions of myosin with actin and 5′-adenylyl imidodiphosphate by substitution of ethylene glycol for water

Biochemical Journal ◽

10.1042/bj2170169 ◽

1984 ◽

Vol 217 (1) ◽

pp. 169-177 ◽

Cited By ~ 13

Author(s):

S B Marston ◽

R T Tregear

Keyword(s):

Ethylene Glycol ◽

Dissociation Rate ◽

Detectable Effect ◽

Muscle Fibres ◽

Skeletal Muscle Myosin ◽

Specific Effects ◽

Myosin Subfragment ◽

Myosin Subfragment 1 ◽

Glycerinated Muscle ◽

Myosin X

We studied the effect of replacing water by ethylene glycol as solvent on the properties of skeletal muscle myosin, myosin subfragment-1 (S1) and heavy meromyosin. Ethylene glycol (50%, v/v) had no detectable effect on the affinity of myosin or actomyosin for the substrate analogue 5′-adenylyl imidodiphosphate (AMPPNP). However, the rate constants for formation and dissociation of the myosin X MgAMPPNP complex were reduced 200-fold; the logarithm of the dissociation rate was roughly proportional to the fractional concentration of ethylene glycol. Nucleotide dissociation was accelerated at least 300-fold by pure actin but remained slow with regulated actin in the absence of Ca2+. Ethylene glycol substitution reduced the affinity of S1 and the S1 X MgAMPPNP complex for actin equally (100-fold at 50% ethylene glycol). These results show that ethylene glycol has specific effects on myosin's enzymic mechanism, which can account for its effect on the tension and stiffness of glycerinated muscle fibres.

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Effect of ethylene glycol on the interaction of different myosin subfragment 1.cntdot.nucleotide complexes with actin

Biochemistry ◽

10.1021/bi00441a048 ◽

1989 ◽

Vol 28 (15) ◽

pp. 6478-6482 ◽

Cited By ~ 2

Author(s):

Ednan Mushtaq ◽

Lois E. Greene

Keyword(s):

Ethylene Glycol ◽

Myosin Subfragment ◽

Myosin Subfragment 1

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Actin-binding compounds that affect the kinetics of the interaction of cardiac myosin with actin

10.1101/2020.09.16.300517 ◽

2020 ◽

Author(s):

Osha Roopnarine ◽

David D. Thomas

Keyword(s):

Steady State ◽

Mechanisms Of Action ◽

Myosin Atpase ◽

New Drugs ◽

Actin Binding ◽

Cardiac Myosin ◽

Myosin Subfragment ◽

Actin Structure ◽

Myosin Subfragment 1 ◽

Kinetics Of

AbstractWe measured the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the microsecond structural dynamics of actin and the steady-state activity of actin-activated myosin ATPase. In the present study, we have further characterized their mechanisms of action by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin, and is restored upon release of S1 by MgATP. We measured the effects of compounds under equilibrium and steady-state conditions, as affected by S1 and ATP, and also under transient conditions, in stopped-flow experiments following rapid addition of ATP to S1-bound PFA. We observe that these compounds affect the early steps of the myosin ATPase cycle to different extents (mild, moderate, and severe). The compounds decrease the equilibrium constant for the formation of the collision complex and the rate constant for subsequent isomerization to the ternary complex, indicating increased ATP affinity and trapping of ATP in the myosin active site. These compound effects on actin structure inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for possible treatment of hypercontractile forms of hypertrophic cardiomyopathy (HCM). This work helps to elucidate the mechanisms of action of these compounds, several of which are currently used therapeutically, and it sets the stage for future HTS campaigns on a larger scale, to discover new drugs for treatment of heart failure.

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Specificity and kinetic effects of nitrophenol analogues that activate myosin subfragment 1

Biochemical Journal ◽

10.1042/bj3240877 ◽

1997 ◽

Vol 324 (3) ◽

pp. 877-884 ◽

Cited By ~ 5

Author(s):

Verônica P. SALERNO ◽

Adriana S. RIBEIRO ◽

Adriana N. DINUCCI ◽

Julio A. MIGNACO ◽

Martha M. SORENSON

Keyword(s):

Mammalian Skeletal Muscle ◽

Head Region ◽

Bivalent Cation ◽

High Substrate ◽

Atp Concentration ◽

Myosin Subfragment ◽

Myosin Subfragment 1 ◽

Kinetic Component ◽

Kinetic Effects ◽

Substrate Concentrations

2,4-Dinitrophenol (DNP) activates the myosin ATPase of mammalian skeletal muscle in the presence of Ca2+ or Mg2+, and inhibits it when the bivalent cations are replaced by K+ and EDTA. Activation of Mg2+ATPase is abolished by the presence of unregulated actin. 3-Nitrophenol (3-NP) is also an activator, whereas other analogues (2-nitrophenol, 2-NP, and 4-nitrophenol, 4-NP) are much less effective. Concentrations required for their half-maximal effects (K0.5) range from 2 to 15 mM for 3-NP and DNP in the presence of different cations, and the sequence for the analogues is 3-NP ⩽ DNP ≪ 2-NP ≈ 4-NP, which is apparently unrelated to either hydrophobicity or pK. DNP and 3-NP have almost identical effects on the ATPase activity of chymotryptic subfragment 1 as they do on myosin, which is an indication that their target is the globular head region rather than the tail, or the 18 kDa (regulatory) light chain. Analysis of the ATP concentration dependence for subfragment-1 ATPase in the presence of Ca2+ or Mg2+ shows that DNP activates only at high substrate concentrations, becoming increasingly effective with ATP concentrations in the physiological range. At low substrate concentrations, DNP inhibits hydrolysis by increasing the apparent Km for ATP at the catalytic site. In the presence of Mg2+, it mimics the effect of actin, which increases the Km and accelerates the release of products following hydrolysis. At high substrate concentrations, activation by DNP appears to involve a kinetic component with low affinity for ATP that can increase the overall reaction rate by a factor of 2- to 9-fold, depending on the bivalent cation. This low-affinity component is either induced by the drug (in the presence of Mg2+) or shifted by the drug to a lower ATP concentration range (in the presence of Ca2+).

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Structure of Myosin Subfragment-1 from Electron Microscopy and Crystallography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102869 ◽

1988 ◽

Vol 46 ◽

pp. 156-157

Author(s):

Donald A. Winkelmann

Keyword(s):

Electron Microscopy ◽

Molecular Weight ◽

Actin Filaments ◽

Light Chains ◽

Myosin Filament ◽

Primary Role ◽

Heavy Chains ◽

Myosin Subfragment ◽

Myosin Subfragment 1 ◽

Globular Head

The primary role of the interaction of actin and myosin is the generation of force and motion as a direct consequence of the cyclic interaction of myosin crossbridges with actin filaments. Myosin is composed of six polypeptides: two heavy chains of molecular weight 220,000 daltons and two pairs of light chains of molecular weight 17,000-23,000. The C-terminal portions of the myosin heavy chains associate to form an α-helical coiled-coil rod which is responsible for myosin filament formation. The N-terminal portion of each heavy chain associates with two different light chains to form a globular head that binds actin and hydrolyses ATP. Myosin can be fragmented by limited proteolysis into several structural and functional domains. It has recently been demonstrated using an in vitro movement assay that the globular head domain, subfragment-1, is sufficient to cause sliding movement of actin filaments.The discovery of conditions for crystallization of the myosin subfragment-1 (S1) has led to a systematic analysis of S1 structure by x-ray crystallography and electron microscopy. Image analysis of electron micrographs of thin sections of small S1 crystals has been used to determine the structure of S1 in the crystal lattice.

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