Terry D R, Spector I, Higa T, Bubb M R
Department of Medicine, Health Science Center, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
J Biol Chem. 1997 Mar 21;272(12):7841-5. doi: 10.1074/jbc.272.12.7841.
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. Analytical ultracentrifugation and steady-state fluorescence experiments show that misakinolide A binds simultaneously to two actin subunits with virtually the same affinity as swinholide A, suggesting that the modification in the ring size does not change the actin-binding site. Sedimentation equilibrium experiments suggest that binding is independent at each binding site, with a Kd of approximately 50 nM. Remarkably, misakinolide A does not sever actin filaments like swinholide A; rather, it caps the barbed end of F-actin. When capped by misakinolide A, the elongation rate constant at the barbed end is reduced to zero; pointed end growth was affected only to the extent that the compound sequesters unpolymerized actin. Misakinolide A has essentially no effect on the off-rate of actin subunits leaving the barbed end. Energy-minimized models of misakinolide A and swinholide A are consistent with conservation 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.
我们研究了海洋天然产物米沙基诺内酯A的生化特性，它是一种由40个原子组成的二聚内酯大环内酯，与斯氏内酯A的区别仅在于大环内酯环的大小。分析超速离心和稳态荧光实验表明，米沙基诺内酯A与两个肌动蛋白亚基同时结合，其亲和力与斯氏内酯A几乎相同，这表明环大小的改变并未改变肌动蛋白结合位点。沉降平衡实验表明，每个结合位点的结合是独立的，解离常数（Kd）约为50 nM。值得注意的是，米沙基诺内酯A不像斯氏内酯A那样切断肌动蛋白丝；相反，它封闭F-肌动蛋白的带刺端。当被米沙基诺内酯A封闭时，带刺端的伸长速率常数降至零；尖端生长仅在该化合物隔离未聚合肌动蛋白的程度上受到影响。米沙基诺内酯A对肌动蛋白亚基离开带刺端的解离速率基本上没有影响。米沙基诺内酯A和斯氏内酯A的能量最小化模型与两个分子中相同结合位点的保守性一致，但一个结合位点相对于另一个结合位点的方向差异可能解释了为什么斯氏内酯A具有切断活性而米沙基诺内酯A仅具有封闭活性。
