Actin Sites Research Articles

Activation of heavy meromyosin adenosine triphosphatase by various states of actin.

F-actin monomer (F-monomer) is formed upon the addition of neutral salt to G-actin. Since F-monomer has a digestibility similar to that of F-actin and much lower than that of G-actin, it has been proposed that F-monomer has a conformation different from that of G-actin and similar to the conformation of the subunits in F-actin. To examine whether F-monomer will enhance the magnesium-activated myosin adenosine triphosphatase (Mg2+-ATPase) as much as F-actin, the ability of partially polymerized actin populations at equilibrium to activate the Mg2+-ATPase of heavy meromyosin was investigated. Correlations were made between ATPase activities and the polymerization state of actin as determined by measurements of viscosity and digestibility. No significant activation of the heavy meromyosin ATPase was observed under conditions where G-actin or mixtures of G-actin and F-monomer were present. As polymer formation occurred at higher actin concentrations, or with increased KCl concentrations, substantial activation characteristic of F-actin was observed. The data suggest that F-monomer may undergo a further conformational change as it forms nuclei or joins onto polymers. Alternatively, the site of actin which activates the myosin ATPase may involve the crevice between two adjacent actin subunits.

The three-state model for the elementary process of energy conversion in muscle

A three-state model for the elementary process of energy conversion in striated muscle is analysed; in two of the three states, the crossbridge is attached to an actin site, while the third is a detached state. This model accounts for the mechanical properties of steady shortening and lengthening processes as well as those of isometric and isotonic transient processes in a quantitative way. Moreover, qualitative agreement is obtained for the total energy liberation from muscle. Biochemical properties are also computed for transient processes. Comparisons are made with other models with “three states”.

Myogenesis and contraction in the early embryonic heart of the rainbow trout

Myogenesis in the embryonic heart of the rainbow trout, Salmo gairdneri (Rich.), was investigated electron microscopically from the 29th to the 41st somite stage. Thick and thin myofilaments are formed simultaneously as well as precursors of Z-lines, to which the thin filaments are attached. The genesis of filaments takes place in the region around the intracellular yolk droplets. The first myofibrils appear by the 33rd somite stage, probably formed by a mechanism of self-assembly in which the binding sites of actin and myosin participate. A- and I-bands do not develop before the 38th osmite stage. The contraction already begins during the 33rd somite stage in the middle of the tubular heart. Gradually, the peristaltic waves spread increasingly to other parts of the heart. In the 41st somite stage the entire heart is contractile and all myocytes contain myofibrils.

Kinetic model of regulation of muscle protein activity

The widely accepted steric model of calcium regulation of actin-myosin interactions in vertebrate muscles has to be completed to fit the kinetic data. It should be supposed that: (1) the thin filaments consist of functionally independent units, containing seven actin sites regulated by one troponin-tropomyosin complex; (2) actin sites become available for myosin heads only due to fluctuations of tropomyosin position; (3) binding of calcium to troponin results either in the shift of the tropomyosin equilibrium position or in the weakening of its interactions with actin strand so that the probability of effective fluctuations increases; (4) link formation between myosin head and some of the available actin site fixates the tropomyosin in such a position that the other six actin sites of the same functional unit become available for myosin too. The model gives linear kinetic scheme for the transitions of a functional unit between nine states (a “turned off” state, and eight “turned on” ones with different occupancy by myosin heads). The dependences of the apparent rate constants of actomyosin formation and dissociation upon the myosin head and substrate concentrations are obtained from the Lymn-Taylor scheme. The frequency of the actomyosin complexes dissociation is assumed to give the ATPase rate. The model fits the kinetic data on the ATP hydrolysis by myosin subfragment-1 with regulated or unregulated actin as a cofactor under various conditions. It shows a sharp dependence of activation upon the apparent affinity of the actin and myosin sites. Therefore, the model appears to be applicable to myosin controlled systems.

Theory of muscular contraction extended to groups of actin sites.

It was shown in an earlier paper how to connect, in principle, the biochemical states of a cross-bridge with the mechanics of muscular contraction, by the methods of statistical mechanics. The treatment applies to cross-bridges that are able to interact with only one actin site at a time. The present paper shows that it is a straightforward matter to extend the theory to groups of actin sites (three, five, etc.), say 55 A apart, as suggested by the work of Moore, H. E. Huxley, and DeRosier. The possibility of the cross-bridge attachment slipping between sites is included. This provides an alternative molecular interpretation of the model introduced by A. F. Huxley and Simmons. A second possible interpretation is also suggested: their discrete stable angles correspond to different biochemical (attached) states. The Huxley-Simmons analysis of an example is rederived and extended somewhat (x averaging), from the point of view of the present theory. Their qualitative conclusions are left unchanged by the x averaging, but significant quantitative effects are possible. Possible consequences of fast slipping in isotonic contraction are discussed in a preliminary way.

Cross-bridge properties derived from muscle isotonic velocity transients.

The rate constants for the turnover of cross-bridges during frog muscle contraction were determined from an analysis of the motion that follows step decreases in load. For a given projection from the myosin filament, there appears to be a range of about 100 A along the length of the filament over which the projection can attach to the actin filament and form a cross-bridge. The site of attachment is then displaced by a distance of this same order before the link is broken. The values of the rate constants also imply that a cross-bridge is formed each time an actin site comes within range of a myosin projection, so that the turnover of cross-bridges for a given contraction distance is independent of the speed of the motion.

Exchange of adenosine diphosphate bound to actin in superprecipitated actomyosin and contracted myofibrils

Up to half of the ADP bound to actin passes into the medium from super-precipitated actomyosin. The presence of ATP or other nucleotide triphosphates is necessary for release to take place. Superprecipitation is also a required condition. If superprecipitation of actomyosin is delayed or inhibited by the omission of magnesium in the presence of the various nucleotide triphosphates or by lowering the temperature, the release is also delayed or inhibited even though ATP or nucleotide triphosphates are present in the medium in great excess. The reaction is an exchange reaction and the actin-bound ADP is replaced by ADP formed from the ATP of the medium. In a system where the actomyosin contains [3H]ADP and the medium [14C]ATP, the loss of [3H]ADP from the actomyosin precipitate is compensated for by the incorporation of [14C]ADP throughout the whole course of the reaction. The same sites can undergo a reaction more than once and a major portion of the newly incorporated [14C]ADP re-exchanges at about the same speed as the unreacted sites containing [3H]ADP. The change on actin leading to the availability of its bound ADP is a transitory one and the ADP newly incorporated into super-precipitated actomyosin cannot serve as cofactor for the creatine kinase-phosphocreatine system. Exchange takes place also on “natural” actomyosin and, though with a slower speed, on washed myofibrils. The exchange reaction with actomyosin starts with a rapid burst, then proceeds at a slower rate. The experiments are interpreted to mean that myosin and ATP in conditions of superprecipitation and contraction impose a change on actin which leads to a loosening of the bond between actin and its bound ADP. The evidence would indicate that repetitive, cyclic reactions occur at the sites of actin and myosin interaction. The rapidly exchanging fraction of ADP may represent the maximum number of actin and myosin sites in contact in an actomyosin precipitate. The slower exchange would be limited by the time required for the completion of the previous cycle and for the unreacted actin sites to come into close proximity with active sites on myosin.†