Actin filaments monomer

Monomeric actin binds ATP very tightly with an association constant Ka of 1 O M in low ionic strength buffers in the presence of Ca ions. A polymerization cycle involves addition of the ATP-monomer to the polymer end, hydrolysis of ATP on the incorporated subunit, liberation of Pi in solution, and dissociation of the ADP-monomer. Exchange of ATP for bound ADP occurs on the monomer only, and precedes its involvement in another polymerization cycle. Therefore, monomer-polymer exchange reactions are linked to the expenditure of energy exactly one mol of ATP per mol of actin is incorporated into actin filaments. As a result, up to 40% of the ATP consumed in motile cells is used to maintain the dynamic state of actin. Thus, it is important to understand how the free energy of nucleotide hydrolysis is utilized in cytoskeleton assembly. 

Polymer growth J(c) showed nonlinear monomer concentration dependence in the presence of ATP (Carrier et al., 1984), while in the presence of ADP, the plot of J(c) versus monomer concentration for actin was a straight line, as expected for reversible polymerization. The data imply that newly incorporated subunits dissociate from the filament at a slower rate than internal ADP-subunits in other words, (a) the effect of nucleotide hydrolysis is to decrease the stability of the polymer by increasing k and (b) nucleotide hydrolysis is uncoupled from polymerization and occurs in a step that follows incorporation of a ATP-subunit in the polymer. Newly incorporated, slowly dissociating, terminal ATP-subunits form a stable cap at the ends of F-actin filaments. 

Pj release occurs at a relatively apparent slow rate (kobs = 0.005 s" ), so that the transient intermediate F-ADP-Pj in which P is non-covalently bound, has a life time of 2-3 minutes (Carlier and Pantaloni, 1986 Carlier, 1987). While the y-phosphate cleavage step is irreversible as assessed by 0 exchange studies (Carlier et al., 1987), the release of Pi is reversible. Binding of H2PO4 (Kp 10 M) causes the stabilization of actin filaments and the rate of filament growth varies linearly with the concentration of actin monomer in the presence of Pi (Carlier and Pantaloni, 1988). Therefore, Pi release appears as the elementary step responsible for the destabilization of actin-actin interactions in the filament. 

In reversible polymerization, the critical concentration is equal to the equilibrium dissociation constant for polymer formation. This parameter is therefore independent of the number of polymers in solution. Confirmation comes from smdying reversible polymerization of ADP-actin when sonic vibration is applied to a solution of F-ADP-actin filaments at equilibrium with G-ADP monomers, no change is observed in the proportion of G- and F-actin (Carlier et al., 1985). Therefore, the only effect of sonic vibration is to increase the number of filaments without affecting the rates of monomer association to and dissociation from filament ends. 

HOW PROFILIN CONTROLS MONOMER-POLYMER STEADY-STATE AND PROMOTES ACTIN FILAMENT ASSEMBLY IN THE PRESENCE OF THYMOSYN p4... 

Actin is a 42 kDa bent dumbbell-shaped globular monomer which is found in most eukaryotic cells. It is the primary protein of the thin (or actin) filaments. Also, by mass or molarity, actin is the largest constituent of the contractile apparatus, actually reaching millimolar concentrations. Actins from different sources seem to be more similar than myosins from the same sources. Actin binds ATP which is hydrolyzed to ADP, if the monomeric actin polymerizes. The backbone structure of the actin filament is a helix formed by two linear strands of polymerized actins like two strings of actin beads entwined. 

Actin filaments are the thinnest of the cytoskeletal filaments, and therefore also called microfilaments. Polymerized actin monomers form long, thin fibers of about 8 nm in diameter. Along with the above-mentioned function of the cytoskeleton, actin interacts with myosin ( thick ) filaments in skeletal muscle fibers to provide the force of muscular contraction. Actin/Myosin interactions also help produce cytoplasmic streaming in most cells. 

Figure 4.1. Actin polymerisation. Actin monomers (G-actin) may reversibly assemble into actin filaments (F-actin). Profilin binds to G actin (to form profilactin) and thus prevents its polymerisation.

Figure 4.4. Structure of actin filaments (a) shows the beads on a string appearance of an actin filament. This filament comprises actin monomers, which themselves have a polarity, so that they can only assemble head to tail , as shown in (b) thus, the actin filament is polarised, having a barbed and a pointed end.

Tropomyosin is a long helical molecule (70 kDa) which extends along the long axis of the actin filament (Figure 13.7). Each tropomyosin molecule covers seven actin monomers and plays a central role in the regulation of muscle contraction. 

Actin filaments grow rapidly within cells, and the clearest evidence of this rapid growth is the ability of the cell s leading edge to move at rates of 0.5 to 1 micrometer per second. Likewise, actin-based motility of Listeria and Shigella can attain rates of nearly 0.5 micrometers per second. Because microfilaments contain about 360 actin monomers per micrometer of length, a motility rate of 0.5 to 1 micrometer per second corresponds to an apparent first-order rate constant (/.e., / apparent = on [Actin-ATP]) of about 180-360 s . The bimolecular rate constant for actin-ATP addition to the barbed end has a nominal value of 2-3 X 10 s . Therefore, one can estimate... 

The mechanical properties of actin filament networks depend on the manner in which actin monomer is prepared and stored, as well as how they are polymerized conditions. Differences in mechanical properties are not the consequence of using two different types of forced oscillatory rheometers. Xu et aid found that filaments assembled in EGTA and Mg from fresh, gel-filtered ATP-actin monomer (1 mg/mL) have an elastic storage... 

The acrosomal process of some invertebrate sperm cells is an actin cable that sometimes forms almost instantaneously by polymerization of the actin monomers and shoots out to penetrate the outer layers of the egg during fertilization (Chapter 32). The stereocilia, the "hairs" of the hair cells in the inner ear, contain bundles of actin filaments.302 Motion of the stereocilia caused by sound produces changes in the membrane potential of the cells initiating a nerve impulse. In certain lizards each hair cell contains about 75 stereocilia of lengths up to 30 pm and diameter 0.8 pm and containing more than 3000 actin filaments in a semicrystalline array. Microvilli (Fig. 1-6) contain longitudinal arrays of actin filaments. 

The two ends of the F-actin filaments have different surfaces of the monomer exposed and grow at different rates. This has been demonstrated by allowing the myosin fragment called heavy meromyosin (HMM see Fig. 19-10) to bind to (or "decorate") an actin filament. The... 

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