bundle, which serves as the nuclei, the orientation of actin filaments is uniform. The
earliest time point (a few seconds after the start of polymerization) depends on how
fast one can prepare the specimen of the electron microscope after mixing the actin
monomer with the nuclei. More recently, the technique for observation of single,
fluorescently-labeled filaments under fluorescence microscope has been developed,
and the rate constant for polymerization/depolymerization kinetics at barbed and
pointed ends of the same filament have been separately measured: it turned out that
the fast-growing and the slow-growing ends were the barbed and the pointed ends
[43]. The rate constants for actin monomer binding ADP, which undergoes reversible polymerization/depolymerization exhibited a remarkable match with those
derived from the optical microscopy (see Sect. 7.4).
7.3.3.2 Actin Polymerization and Hydrolysis of ATP
Actin monomer is an ATPase, as mentioned above. It hydrolyzes ATP only once at a
rate of ~0.02/sec when it is incorporated into the filament [44]; the product, inorganic
phosphate (Pi), dissociates from the filament at ~0.3/s [45]. The other product, ADP,
remains bound to the actin protomer; but when the protomer dissociates from actin
filament, the ADP bound to the dissociated monomer can readily exchange with
ATP in the surrounding medium. If actin monomer loses bound nucleotide, it
denatures and becomes polymerization incompetent [46]. Monomer without bound
nucleotide is polymerization competent, if a high concentration of sucrose or
glycerol is present [46, 47].
In the presence of ATP, the actin monomer and actin filament are in a
non-equilibrium steady state because of the ATPase activity of actin (see Sect.
7.5). As mentioned above, the ATP hydrolysis occurs at a finite rate, and hence, if
monomer concentration is high, the rate of association of monomer will surpass the
rate of ATP hydrolysis, and as a result, several protomers near the barbed end can
exist as ATP-bound form [45, 48]. The dissociation of inorganic phosphate from the
actin protomer at the filament ends is followed by dissociation of this ADP-binding
protomer [43]; in the protomers residing in the middle of the filament, dissociation of
inorganic phosphate is 1000 times slower than the filament end [43, 49]. Phosphate
binds to actin protomer with a dissociation constant of 1.5 mM [50]. The bound Pi
decreases the dissociation rate of the monomer that binds ADP, leading to a decrease
in the critical concentration of the barbed and the pointed ends [43, 51]. In in vitro
experiment, Pi is often omitted, but in vivo, Pi exists at millimolar concentrations,
which will considerably increase the stability of the actin filament against depolymerization [43]. The effect of the type of nucleotide at the ends of the filaments in the
nuclei does not seem to be reflected in the elongation rate [42]. This fact simplifies
the comparison of the data obtained with the electron microscopy and fluorescence
microscopy.
7.3 Actin
111
earliest time point (a few seconds after the start of polymerization) depends on how
fast one can prepare the specimen of the electron microscope after mixing the actin
monomer with the nuclei. More recently, the technique for observation of single,
fluorescently-labeled filaments under fluorescence microscope has been developed,
and the rate constant for polymerization/depolymerization kinetics at barbed and
pointed ends of the same filament have been separately measured: it turned out that
the fast-growing and the slow-growing ends were the barbed and the pointed ends
[43]. The rate constants for actin monomer binding ADP, which undergoes reversible polymerization/depolymerization exhibited a remarkable match with those
derived from the optical microscopy (see Sect. 7.4).
7.3.3.2 Actin Polymerization and Hydrolysis of ATP
Actin monomer is an ATPase, as mentioned above. It hydrolyzes ATP only once at a
rate of ~0.02/sec when it is incorporated into the filament [44]; the product, inorganic
phosphate (Pi), dissociates from the filament at ~0.3/s [45]. The other product, ADP,
remains bound to the actin protomer; but when the protomer dissociates from actin
filament, the ADP bound to the dissociated monomer can readily exchange with
ATP in the surrounding medium. If actin monomer loses bound nucleotide, it
denatures and becomes polymerization incompetent [46]. Monomer without bound
nucleotide is polymerization competent, if a high concentration of sucrose or
glycerol is present [46, 47].
In the presence of ATP, the actin monomer and actin filament are in a
non-equilibrium steady state because of the ATPase activity of actin (see Sect.
7.5). As mentioned above, the ATP hydrolysis occurs at a finite rate, and hence, if
monomer concentration is high, the rate of association of monomer will surpass the
rate of ATP hydrolysis, and as a result, several protomers near the barbed end can
exist as ATP-bound form [45, 48]. The dissociation of inorganic phosphate from the
actin protomer at the filament ends is followed by dissociation of this ADP-binding
protomer [43]; in the protomers residing in the middle of the filament, dissociation of
inorganic phosphate is 1000 times slower than the filament end [43, 49]. Phosphate
binds to actin protomer with a dissociation constant of 1.5 mM [50]. The bound Pi
decreases the dissociation rate of the monomer that binds ADP, leading to a decrease
in the critical concentration of the barbed and the pointed ends [43, 51]. In in vitro
experiment, Pi is often omitted, but in vivo, Pi exists at millimolar concentrations,
which will considerably increase the stability of the actin filament against depolymerization [43]. The effect of the type of nucleotide at the ends of the filaments in the
nuclei does not seem to be reflected in the elongation rate [42]. This fact simplifies
the comparison of the data obtained with the electron microscopy and fluorescence
microscopy.
7.3 Actin
111