7.3.3.3 Structure of the Barbed and the Pointed Ends
and Polymerization/Depolymerization
The difference in the polymerization/depolymerization kinetics of actin filament has
been interpreted on the basis of the structure of the filament ends [33]. The actin
filaments were prepared in the presence of a cap Z protein, which binds to the barbed
end of an actin filament and stabilize it against depolymerization. This treatment
increases the number of filament with free pointed ends. With this preparation, it was
found that at the pointed end, the terminal protomer slightly tilted toward the
immediate next protomer and an interaction occurred between two amino-acid
loops that extend from each protomer [33]. It has been postulated that this looploop interaction creates a kinetic barrier against the association and the dissociation
of the terminal protomer, thereby lowering the rate constants of association and the
dissociation. Such strong inter-protomer interaction is not observed at the barbed
end, and hence, the rates of dissociation and association of the terminal protomer at
the barbed end will be higher than those of the terminal protomer at the pointed end.
The slow association of the protomer at the pointed end will lead to the synchronization of ATP hydrolysis with the association of the monomer. On the other hand, as
described above, the ATP hydrolysis end lags behind the association of monomer at
the barbed, if concentration of monomer is sufficiently high.
7.3.3.4 Actin Polymerization and Metal Ions
In vitro, the purified actin polymerizes in the presence of cations (K
+
, Mg
2+ or Ca
2+
ions). As described above, actin monomer possesses one high affinity binding site
for Mg
2+ /Ca
2+ ions near the ATP binding site and several binding sites for cations
with lower affinity [27]. Upon binding of monovalent cations to these low affinity
sites, the electrostatic repulsion between monomers will decrease, facilitating the
association of monomer to each other. Divalent cations induce polymerization at
much lower concentration than monovalent cations; to polymerize actin, 1 mM Mg
2+ is sufficient, whereas K
+ ions of tens of mM are required. Under the polymerization condition that is usually adopted in in vitro experiment (a few micromolar of
monomer with 1 mM Mg
2+ or with 1 mM Mg
2+ and 10–100 mM K
+ ions),
polymerization occurs on the order of 10 min. Conversely, if monomer concentration is high, even a submillimolar concentration of Ca
2+ causes polymerization on a
time scale of scores of minutes [36].
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7 Moving Life
and Polymerization/Depolymerization
The difference in the polymerization/depolymerization kinetics of actin filament has
been interpreted on the basis of the structure of the filament ends [33]. The actin
filaments were prepared in the presence of a cap Z protein, which binds to the barbed
end of an actin filament and stabilize it against depolymerization. This treatment
increases the number of filament with free pointed ends. With this preparation, it was
found that at the pointed end, the terminal protomer slightly tilted toward the
immediate next protomer and an interaction occurred between two amino-acid
loops that extend from each protomer [33]. It has been postulated that this looploop interaction creates a kinetic barrier against the association and the dissociation
of the terminal protomer, thereby lowering the rate constants of association and the
dissociation. Such strong inter-protomer interaction is not observed at the barbed
end, and hence, the rates of dissociation and association of the terminal protomer at
the barbed end will be higher than those of the terminal protomer at the pointed end.
The slow association of the protomer at the pointed end will lead to the synchronization of ATP hydrolysis with the association of the monomer. On the other hand, as
described above, the ATP hydrolysis end lags behind the association of monomer at
the barbed, if concentration of monomer is sufficiently high.
7.3.3.4 Actin Polymerization and Metal Ions
In vitro, the purified actin polymerizes in the presence of cations (K
+
, Mg
2+ or Ca
2+
ions). As described above, actin monomer possesses one high affinity binding site
for Mg
2+ /Ca
2+ ions near the ATP binding site and several binding sites for cations
with lower affinity [27]. Upon binding of monovalent cations to these low affinity
sites, the electrostatic repulsion between monomers will decrease, facilitating the
association of monomer to each other. Divalent cations induce polymerization at
much lower concentration than monovalent cations; to polymerize actin, 1 mM Mg
2+ is sufficient, whereas K
+ ions of tens of mM are required. Under the polymerization condition that is usually adopted in in vitro experiment (a few micromolar of
monomer with 1 mM Mg
2+ or with 1 mM Mg
2+ and 10–100 mM K
+ ions),
polymerization occurs on the order of 10 min. Conversely, if monomer concentration is high, even a submillimolar concentration of Ca
2+ causes polymerization on a
time scale of scores of minutes [36].
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7 Moving Life