Q

- Nucleation -

Nuclei added

Nucleation

Elongation

Nucleus

F-actin

Elongation

Nucleus

Steady state

F-actin

Steady state

Nuclei added

Time

▲ EXPERIMENTAL FIGURE 19-6 Polymerization of G-actin in vitro occurs in three phases. (a) In the Initial nucleation phase, ATP-G-actin monomers (pink) slowly form stable complexes of actin (purple). These nuclei are rapidly elongated in the second phase by the addition of subunits to both ends of the filament. In the third phase, the ends of actin filaments are in a steady state with monomeric ATP-G-actin. After their incorporation into a filament, subunits slowly hydrolyze ATP and become stable ADP-F-actin (white). Note that the ATP-binding clefts of all the subunits are oriented in the same direction in F-actin. (b) Time course of the in vitro polymerization reaction (pink curve) reveals the initial lag period. If some actin filament fragments are added at the start of the reaction to act as nuclei, elongation proceeds immediately without any lag period (purple curve).

Cc Actin concentration

▲ EXPERIMENTAL FIGURE 19-7 Concentration of G-actin determines filament formation. The critical concentration (Cc) is the concentration of G-actin monomers in equilibrium with actin filaments. At monomer concentrations below the Cc, no polymerization takes place. At monomer concentrations above the Cc, filaments assemble until the monomer concentration tain length (three or four subunits), it can act as a stable seed, or nucleus, which in the second elongation phase rapidly increases in length by the addition of actin monomers to both of its ends. As F-actin filaments grow, the concentration of G-actin monomers decreases until equilibrium is reached between filaments and monomers. In this third steady-state phase, G-actin monomers exchange with subunits at the filament ends, but there is no net change in the total mass of filaments. The kinetic curves presented in Figure 19-6b show that the lag period can be eliminated by the addition of a small number of F-actin nuclei to the solution of G-actin.

When the steady-state phase has been reached, the concentration of the pool of unassembled subunits is called the critical concentration, Cc. This parameter is the dissociation constant, the ratio of the "on" and "off" rate constants, and it measures the concentration of G-actin where the addition of subunits is balanced by the dissociation of subunits; that is, the on rate equals the off rate. Under typical in vitro conditions, the Cc of G-actin is 0.1 ^M. Above this value, a solution of G-actin will polymerize; below this value, a solution of F-actin will depolymerize (Figure 19-7).

After ATP-G-actin monomers are incorporated into a filament, the bound ATP is slowly hydrolyzed to ADP. As a result of this hydrolysis, most of the filament consists of ADP-F-actin, but some ATP-F-actin is found at one end (see next subsection). However, ATP hydrolysis is not essential for polymerization to take place, as evidenced by the ability of G-actin containing ADP or a nonhydrolyzable ATP analog to polymerize into filaments.

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