1: J Biochem (Tokyo)  1997 Mar;121(3):527-33 

Cooperative association of actin protomers and crosslinked actin oligomers in
filaments at low ionic strength.

Miyata H, Kinosita K Jr, Marriott G.

Department of Physics, Faculty of Science and Technology, Keio University,
Kohoku-ku, Yokohama. miyata@bio.phys.tohoku.ac.jp

F-Actin treated with a half molar equivalent of the heterobifunctional
cross-linker, 4-bromomethyl-3-nitrobenzoic acid succinimide ester (BNBA-SE),
produced a population of actin oligomers containing 2-14 or more protomers and a
significant amount of uncrosslinked actin protomers. The crosslinking reaction
is presumed to occur between Cys 374 of one actin protomer with Lys 191 of an
adjacent protomer on the genetic helix of F-actin, in a similar manner to
N-maleimidobenzoic acid succinimidyl ester, which shares similar reactive groups
and crosslinking dimensions. Crosslinked oligomers and uncrosslinked protomers
were found to form filaments that sediment after high-speed centrifugation, even
in a buffer of low ionic strength [G-buffer: 2 mM tris-hydroxymethylaminomethane
(pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.3 mM NaN3, 5 mM 2-mercaptoethanol] where
affinity between actin protomers usually becomes weak, leading to the
depolymerization of native F-actin. By performing the crosslinking reaction in
the presence of an environment-sensitive fluorescent label,
6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), which competes with BNBA-SE
for Cys 374 of actin protomers, fluorescent, crosslinked F-actin filaments were
obtained which were also stable in G-buffer. Fluorometric analysis of actin
labeled with acrylodan (prodan-actin) in these depolymerization-resistant
filaments suggested that the molecular environment around Cys 374 of actin
protomers is similar to that of actin protomers in native F-actin in F-buffer
(G-buffer plus 0.1 M KCl and 1 mM MgCl2). When G-actin labeled with acrylodan
was co-polymerized with non-fluorescent crosslinked F-actin, some of the labeled
actin was incorporated into filaments that were resistant to depolymerization in
G-buffer. The emission spectrum of the prodan-actin in these filaments measured
in G-buffer was almost identical to that of prodan-actin in native F-actin in
F-buffer. Our interpretation of this result is that actin protomers are locked
into an F-actin-like conformation in the depolymerization-resistant filament by
the subunit interactions they make with crosslinked oligomers. We also present
evidence which suggests that the depolymerization rate in G-buffer of filaments
made with crosslinked oligomers is much slower than that of native actin because
the ends of the depolymerization-resistant filaments are capped with crosslinked
oligomers.

PMID: 9133622 [PubMed - indexed for MEDLINE]