E. Kim, C. Miller, A. Bobkova, A. Muhlrad, G. Hegyi and E. Reisler
Department of of Chemistry and Biochemistry and Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095.
4-azido-2-nitrophenyl-putrescine attached to Q41 on actin, as described by (Hegyi et al., Protein Sci., 1992, 1, 132-144), was crosslinked to adjacent actin units along the two start helix by photoactivation. SDS PAGE analysis of the reacted actin revealed a ladder of crosslinked dimers, trimers, and higher oligomers. The crosslinking had no effect on the rigor binding of S1 to actin and did not change significantly the Vmax and Km values of acto-S1 ATPase. Fractionation of depolymerized crosslinked actin yielded three fractions: dimers, trimers(+dimers), and oligomers. These crosslinked actins activated S1 ATPase only up to three-fold better then G-actin, despite their strong binding of S1. Crosslinked actin dimers appeared to bind the first S1 much stronger and the second one at a comparable affinity to that of G-actin. The three fractions of crosslinked actin were readily polymerized to F-actin by MgCl2. After repolymerization, the filamentous, crosslinked actin activated the S1 ATPase to about 50% of the activation by control F-actin. In vitro motilities of the filaments were inhibited by the crosslinking of the filamentous actin. The motility of filaments reconstituted from the crosslinked dimers, trimers, and oligomers was inhibited by 40, 75, and 100%, respectively. These results support the claim on the active role of actin in mechanochemical events.