MISAKINOLIDE-A IS A MARINE MACROLIDE THAT CAPS BUT DOES NOT SEVER FILAMENTOUS ACTIN

We have investigated the biochemical properties of the marine natural product, misakinolide A, a 40-membered dimeric lactone macrolide that differs from swinholide A only in the size of the macrolide ring, Anal ytical ultracentrifugation and steady-state fluorescence experiments s how that misakinolide A binds simultaneously to two actin subunits wit h virtually the same affinity as swinholide A, suggesting that the mod ification in the ring size does not change the actin-binding site, Sed imentation equilibrium experiments suggest that binding is independent at each binding site, with a K-d of approximately 50 nM, Remarkably, misakinolide A does not sever actin filaments like swinholide A; rathe r, it caps the barbed end of F-actin, When capped by misakinolide A, t he elongation rate constant at the barbed end is reduced to zero; poin ted end growth was affected only to the extent that the compound seque sters unpolymerized actin, Misakinolide A has essentially no effect on the off-rate of actin subunits leaving the barbed end, Energy-minimiz ed models of misakinolide A and swinholide A are consistent with conse rvation of identical binding sites in both molecules, but a difference in orientation of one binding site relative to the other may explain why swinholide A has severing activity whereas misakinolide A only has capping activity.