Kinetic studies on the initial contraction dependent high ATPase activity of actomyosin molecules

In contracting (superprecipitating) clearing and fully contracted (previously superprecipitated) actomyosin molecules the presteady state phosphate burst was found to be 2 nanomoles inorganic phosphate (Pi) per nanomole myosin. In these muscle models a significant difference in the Mg2+ ATPase activity was found following the initial phosphate burst. Between 120 and 800 milliseconds after the commencement of the reaction the Mg2+ ATPase activity of contracting actomyosin molecules was 5-10 times greater than that of the fully contracted or clearing actomyosin molecules. In the same time interval the rate of turbidity increase of the contracting actomyosin molecules was about 10 fold greater than during the remainder of the time to reach maximal superprecipitation. This high initial ATPase activity found to be present only in the contracting actomyosin molecules and coinciding with the high rate of the velocity of contraction provides sufficient energy for contraction. We propose that this high Mg2+--ATPase activity following the initial burst and included as a part of "conventional" steady state ATPase activity is the source of energy for muscular contraction. Calculation of kinetic and thermodynamic constants indicates that the contracting actomyosin molecule is subjected to a conformational change. As a consequence of contraction the complementarity of the enzyme site to the intermediate complex decreases about 100 fold. Thus the contracted molecules temporarily become relatively refractive to provide energy for the contractile process. In our opinion these findings are important with regard to muscular contraction.     

Effect of Nucleotides on the Orientation and Mobility of Myosin Subfragment-1 in Ghost Muscle Fiber

Using polarization fluorimetry, the orientation and mobility of 1,5-IAEDANS specifically bound to Cys707 of myosin subfragment-1 (S1) were studied in ghost muscle tropomyosin-containing fibers in the absence and in the presence of MgADP, MgAMP-PNP, MgATPgammaS, or MgATP. Modeling of various intermediate states was accompanied by discrete changes in actomyosin orientation and mobility of fluorescent dye dipoles. This suggests multistep changes in the structural state of the myosin head during the ATPase cycle. Maximal differences in the probe orientation by 4 degrees and its mobility by 30% were found between actomyosin states in the presence of MgADP and MgATP. It is suggested that interaction of S1 with F-actin induces nucleotide-dependent rotation of the whole motor domain of the myosin head or only the dye-binding site and also change in the head mobility.     

The force exerted by a muscle cross-bridge depends directly on the strength of the actomyosin bond

Myosin produces force in a cyclic interaction, which involves alternate tight binding to actin and to ATP. We have investigated the energetics associated with force production by measuring the force generated by skinned muscle fibers as the strength of the actomyosin bond is changed. We varied the strength of the actomyosin bond by addition of a polymer that promotes protein-protein association or by changing temperature or ionic strength. We estimated the free energy available to generate force by measuring isometric tension, as the free energy of the states that precede the working stroke are lowered with increasing phosphate. We found that the free energy available to generate force and the force per attached cross-bridge at low [Pi] were both proportional to the free energy available from the formation of the actomyosin bond. We conclude that the formation of the actomyosin bond is involved in providing the free energy driving the production of isometric tension and mechanical work. Because the binding of myosin to actin is an endothermic, entropically driven reaction, work must be performed by a "thermal ratchet" in which a thermal fluctuation in Brownian motion is captured by formation of the actomyosin bond.     

A possible role of myosin conformers in provision of dual-stage superprecipitation kinetics of natural actomyosin

It has been shown that synchronous starting and successive accomplishment of superprecipitation on the two types of actomyosin complexes lead to the two-stage kinetics of this reaction. By means of a temperature change different balance of two types of actomyosin macromolecules can be achieved. We conclude that two different structural forms (conformers) of myosin cause two non-equivalent functional states of the whole actomyosin complex.     

A molecular mechanism for the production of muscular work

Motility in biological systems is widely thought to result from the transduction of chemical free energy. In muscle a difficulty has been encountered in finding a precise mechanism whereby this conversion is accomplished. We suggest that this difficulty resides in the macroscopic character of free energy which, as a thermodynamic quantity, deals only with large assemblages of molecules. However, the fundamental site of active movement has recently been found to be localized in a single molecule (a myosin head) and is therefore not open to thermodynamic treatment. It is suggested instead that the energetic source of work produced at the myosin head is to be found in the heat (in the form of kinetic energy) evolved during an actomyosin ATPase cycle. This heat equivalent kinetic energy is then converted into useful work by means of a vibrational mode of a single water molecule which is attached to the ADP formed in the myosin head during a portion of the actomyosin ATPase cycle. It is the resonance mode of this water molecule which enables the extremely short durations (10^-15s) of the chemical reactions taking place in one actomyosin ATPase cycle to result in the much longer duration (10^-2s) of the resulting movement. This mechanism may also be fundamental to other types of motility in living systems.     

The synergetic enzyme theory of muscular contraction: II Relation between Hill's equations and functions of two-headed myosin.

Based on the assumption of nonidentical two heads of myosin it is pointed out that a strong motive force is generated in actomyosin pair only when ATP-decomposition occurs co-operatively at the both heads and that the tension-independent part of shortening heat is liberated when an ATP molecule is decomposed only at the burst head. These two actions of actomyosin pair are related to the two states of force-generator in Huxley-Simmons' model. Elementary cycles at different positions in a sarcomere are interacted each other through feedback loop via sliding motion of muscular filaments. Due to this synergetic interaction the rate constant for the rate-determining step of elementary cycle has a dependence on velocity v of shortening such as k = k ° + κv. From these functions and properties of actomyosin system in vivo, the following properties of muscle are explained consistently in a quantitative manner: (1) Hill's equation on the relationship between tension and velocity of shortening, (2) damped oscillations in tension and in muscular length around steady state, (3) Hill's energy equation improved in 1964, (4) the chemical equivalence of shortening heat, (5) the influence of tension on the incorporation of radio-active phosphate into ATP and (6) the asymmetric activation by actomyosin system only for the forward reaction, the decomposition of ATP.     

A unique ATP hydrolysis mechanism of single-headed processive myosin, myosin IX

Recent studies have revealed that myosin IX is a single-headed processive myosin, yet it is unclear how myosin IX can achieve the processive movement. Here we studied the mechanism of ATP hydrolysis cycle of actomyosin IXb. We found that myosin IXb has a rate-limiting ATP hydrolysis step unlike other known myosins, thus populating the prehydrolysis intermediate (M.ATP). M.ATP has a high affinity for actin, and, unlike other myosins, the dissociation of M.ATP from actin was extremely slow, thus preventing myosin from dissociating away from actin. The ADP dissociation step was 10-fold faster than the overall ATP hydrolysis cycle rate and thus not rate-limiting. We propose the following model for single-headed processive myosin. Upon the formation of the M.ATP intermediate, the tight binding of actomyosin IX at the interface is weakened. However, the head is kept in close proximity to actin due to the tethering role of loop 2/large unique insertion of myosin IX. There is enough freedom for the myosin head to find the next location of the binding site along with the actin filament before complete dissociation from the filament. After ATP hydrolysis, Pi is quickly released to form a strong actin binding form, and a power stroke takes place.     

Superprecipitation of actomyosin with p-chloromercuribenzoate-modified myosin reconstituted from rabbit skeletal muscle

Myosin head modified with p-chloromercuribenzoate (CMB) forms rigor-like complex with actin in the presence of ATP. Actomyosins with CMB-modified myosin were reconstituted to study the effect of rigor-like complexes on superprecipitation. As native myosin was increasingly replaced by CMB-modified myosin, superprecipitation of the actomyosin was strongly suppressed. Further, the suppression of superprecipitation occurred in a different fashion depending on how CMB-modified myosin was incorporated in myosin filaments of the reconstituted actomyosin. The present results indicate that superprecipitation requires the dissociation of actin and myosin head to take place (i.e., the presence of molecular rearrangements of actomyosin network), and further suggest that superprecipitation is associated with dynamic rearrangements of actomyosin network along myosin filaments.     

Myosin structure: does the tail wag the dog?

Previous crystal structures of the myosin head have shown two different conformations, postulated to be the beginning and the end of the actomyosin power stroke. A new crystal structure reveals a dramatically different conformation; but how does this conformation fit into the force-generating cycle of actomyosin interactions?     

Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.     

Reaction of two heads of gizzard myosin with ATP

The reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MPADP, formed was measured from the Pi-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the Pi-burst size was 0.5 mol/mol myosin head, and the apparent Kd value was about 0.15 microM. However, at high ATP concentrations, the Pi burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 microM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd congruent to 1 microM) and 0.35 mol of nucleotides with low affinity (Kd = 24 microM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MPADP with high affinity for ATP while the other forms MPADP and MATP with low affinity for ATP. We studied the correlation between the formation of MPADP and the dissociation of actomyosin. The amount of Pi-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1 mg/ml F-actin, 5 microM myosin at 0 degrees C) most (93%) of the added ATP was hydrolyzed in the Pi-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The evolutionary pathway from anoxygenic to oxygenic photosynthesis examined by comparison of the properties of photosystem II and bacterial reaction centers

In photosynthetic organisms, such as purple bacteria, cyanobacteria, and plants, light is captured and converted into energy to create energy-rich compounds. The primary process of energy conversion involves the transfer of electrons from an excited donor molecule to a series of electron acceptors in pigment–protein complexes. Two of these complexes, the bacterial reaction center and photosystem II, are evolutionarily related and structurally similar. However, only photosystem II is capable of performing the unique reaction of water oxidation. An understanding of the evolutionary process that lead to the development of oxygenic photosynthesis can be found by comparison of these two complexes. In this review, we summarize how insight is being gained by examination of the differences in critical functional properties of these complexes and by experimental efforts to alter pigment–protein interactions of the bacterial reaction center in order to enable it to perform reactions, such as amino acid and metal oxidation, observable in photosystem II.     

A model of muscle contraction based on the Langevin equation with actomyosin potentials

We propose a muscle contraction model that is essentially a model of the motion of myosin motors as described by a Langevin equation. This model involves one-dimensional numerical calculations wherein the total force is the sum of a viscous force proportional to the myosin head velocity, a white Gaussian noise produced by random forces and other potential forces originating from the actomyosin structure and intra-molecular charges. We calculate the velocity of a single myosin on an actin filament to be 4.9–49 μm/s, depending on the viscosity between the actomyosin molecules. A myosin filament with a hundred myosin heads is used to simulate the contractions of a half-sarcomere within the skeletal muscle. The force response due to a quick release in the isometric contraction is simulated using a process wherein crossbridges are changed forcibly from one state to another. In contrast, the force response to a quick stretch is simulated using purely mechanical characteristics. We simulate the force–velocity relation and energy efficiency in the isotonic contraction and adenosine triphosphate consumption. The simulation results are in good agreement with the experimental results. We show that the Langevin equation for the actomyosin potentials can be modified statistically to become an existing muscle model that uses Maxwell elements.     

The two pathways for oxygen exchange by actomyosin and myofibrils and their dependence on temperature

At an intermediate stage in the hydrolysis of MgATP by actomyosin there is an exchange of oxygen between water and the terminal phosphoryl group of MgATP, tightly bound to the myosin active site. This intermediate oxygen exchange results from the reversible hydrolysis of the bound MgATP. The rate of the exchange cycle (hydrolysis and the reverse) is assumed to be determined by the rate of reverse hydrolysis; and the average time available for exchange is determined by the post-exchange reaction that immediately follows the cycle. Past analytical studies of the exchange, using actomyosin mixtures and myofibrils at room temperature, have revealed two pathways for hydrolysis, operating at a comparable flux but differing greatly in the extent of exchange they support. It is shown here that these pathways also appear over a range of temperatures from 5 to 30 degrees C and that temperature had little effect on their relative fluxes. At each temperature, the flux ratio (%) for the low exchange pathway: high exchange pathway was near 50:50 for actomyosin mixtures and 60:40 for myofibrils. Apparently, the rate-limiting steps that determine the fluxes of the two pathways have a similar temperature dependence. However, the analysis indicates that one or both of the steps that determine the extent of exchange (reverse-hydrolysis and/or the post-exchange reaction) shows a different temperature dependence for the two pathways. We interpret this to reflect a difference in the temperature dependence of the post-exchange reaction, which we propose is exceedingly fast and independent of actin concentration along the low exchange route, but slow and dependent on the actin concentration along the high exchange route. Thus at all temperatures over a broad range of actin concentration there are two pathways of comparable flux that differ primarily in the time available for exchange.     

pH-dependent changes in the two-stage kinetics of superprecipitation reaction of natural actomyosin

The effect of pH on the two-stage kinetics of the superprecipitation (SPP) reaction of natural actomyosin was investigated. It was shown that the experimental dependencies appear as two intersecting bell-shaped curves reflecting the effects of pH on individual steps of the SPP reaction which are mediated by different molecular mechanisms. It was supposed that the both reaction mechanisms involve actomyosin complexes which have different structural states and differ also by the degree of dissociation in the presence of ATP. The shifts in the dynamic equilibrium between the two states of actomyosin may induce pH-modulations in the two-stage kinetics of SPP and, presumably, ATPase.     

The role of transporters in supplying energy to plant plastids

The energy status of plant cells strongly depends on the energy metabolism in chloroplasts and mitochondria, which are capable of generating ATP either by photosynthetic or oxidative phosphorylation, respectively. Another energy-rich metabolite inside plastids is the glycolytic intermediate phosphoenolpyruvate (PEP). However, chloroplasts and most non-green plastids lack the ability to generate PEP via a complete glycolytic pathway. Hence, PEP import mediated by the plastidic PEP/phosphate translocator or PEP provided by the plastidic enolase are vital for plant growth and development. In contrast to chloroplasts, metabolism in non-green plastids (amyloplasts) of starch-storing tissues strongly depends on both the import of ATP mediated by the plastidic nucleotide transporter NTT and of carbon (glucose 6-phosphate, Glc6P) mediated by the plastidic Glc6P/phosphate translocator (GPT). Both transporters have been shown to co-limit starch biosynthesis in potato plants. In addition, non-photosynthetic plastids as well as chloroplasts during the night rely on the import of energy in the form of ATP via the NTT. During energy starvation such as prolonged darkness, chloroplasts strongly depend on the supply of ATP which can be provided by lipid respiration, a process involving chloroplasts, peroxisomes, and mitochondria and the transport of intermediates, i.e. fatty acids, ATP, citrate, and oxaloacetate across their membranes. The role of transporters involved in the provision of energy-rich metabolites and in pathways supplying plastids with metabolic energy is summarized here.     

Electric dipole theory and thermodynamics of actomyosin molecular motor in muscle contraction

Movements in muscles are generated by the myosins which interact with the actin filaments. In this paper we present an electric theory to describe how the chemical energy is first stored in electrostatic form in the myosin system and how it is then released and transformed into work. Due to the longitudinal polarized molecular structure with the negative phosphate group tail, the ATP molecule possesses a large electric dipole moment (p(0)), which makes it an ideal energy source for the electric dipole motor of the actomyosin system. The myosin head contains a large number of strongly restrained water molecules, which makes the ATP-driven electric dipole motor possible. The strongly restrained water molecules can store the chemical energy released by ATP binding and hydrolysis processes in the electric form due to their myosin structure fixed electric dipole moments (p(i)). The decrease in the electric energy is transformed into mechanical work by the rotational movement of the myosin head, which follows from the interaction of the dipoles p(i) with the potential field V(0) of ATP and with the potential field Psi of the actin. The electrical meaning of the hydrolysis reaction is to reduce the dipole moment p(0)-the remaining dipole moment of the adenosine diphosphate (ADP) is appropriately smaller to return the low negative value of the electric energy nearly back to its initial value, enabling the removal of ADP from the myosin head so that the cycling process can be repeated. We derive for the electric energy of the myosin system a general equation, which contains the potential field V(0) with the dipole moment p(0), the dipole moments p(i) and the potential field psi. Using the previously published experimental data for the electric dipole of ATP (p(0) congruent with 230 debye) and for the amount of strongly restrained water molecules (N congruent with 720) in the myosin subfragment (S1), we show that the Gibbs free energy changes of the ATP binding and hydrolysis reaction steps can be converted into the form of electric energy. The mechanical action between myosin and actin is investigated by the principle of virtual work. An electric torque always appears, i.e. a moment of electric forces between dipoles p(0) and p(i)(/M/ > or = 16 pN nm) that causes the myosin head to function like a scissors-shaped electric dipole motor. The theory as a whole is illustrated by several numerical examples and the results are compared with experimental results.     

Mechanism of coupling of oxidation and phosphorylation

The mechanism of the energy coupling process proposed in the present investigation is based on energy transformation with participation of chemical intermediates, paramagnetic molecules formed in unsaturated fatty acid chains of the phospholipid membrane. The proposed mechanism is a modification of the chemical-intermediate hypothesis, with energy-rich lipid radicals serving as an intermediate. Although there are some points of inconsistency with the chemiosmic theory and Williams's hypothesis about the energy-rich proton, the proposed mechanism accounts for the delta mu H+ gradient and intramembrane proton during the coupling process in oxidative phosphorylation. The existence of the delta mu H+ gradient and its variation during phosphorylation may be a way of oxidative control realization.     

The Role of Energy in the Emergence of Biology from Chemistry

Any scenario of the transition from chemistry to biology should include an ??energy module?? because life can exist only when supported by energy flow(s). We addressed the problem of primordial energetics by combining physico-chemical considerations with phylogenomic analysis. We propose that the first replicators could use abiotically formed, exceptionally photostable activated cyclic nucleotides both as building blocks and as the main energy source. Nucleoside triphosphates could replace cyclic nucleotides as the principal energy-rich compounds at the stage of the first cells, presumably because the metal chelates of nucleoside triphosphates penetrated membranes much better than the respective metal complexes of nucleoside monophosphates. The ability to exploit natural energy flows for biogenic production of energy-rich molecules could evolve only gradually, after the emergence of sophisticated enzymes and ion-tight membranes. We argue that, in the course of evolution, sodium-dependent membrane energetics preceded the proton-based energetics which evolved independently in bacteria and archaea.     

Mechanism of action of myosin X, a membrane-associated molecular motor

We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.