De La Cruz E M, Pollard T D
Department of Cell Biology & Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Biochemistry. 1996 Nov 12;35(45):14054-61. doi: 10.1021/bi961047t.
We compared the kinetics and thermodynamics of rhodamine phalloidin binding to actin purified from rabbit skeletal muscle, Acanthamoeba castellanii, and Saccharomyces cerevisiae in 50 mM KCl, 1 mM MgCl2, and pH 7.0 buffer at 22 degrees C. Filaments of S. cerevisiae actin bind rhodamine phalloidin more weakly than Acanthamoeba and rabbit skeletal muscle actin filaments due to a more rapid dissociation rate in spite of a significantly faster association rate constant. The higher dissociation rate constant and lower binding affinity of rhodamine phalloidin for S. cerevisiae actin filaments provide a quantitative explanation for the inefficient staining of yeast actin filaments, compared with that of rabbit skeletal muscle actin filaments [Kron et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4466-4470]. The temperature dependence of the rate constants was interpreted according to transition state theory. There is a small enthalpic difference (delta H++) between the ground states and the transition state. Consequently, the free energy of activation (delta G++) for association and dissociation of rhodamine phalloidin is dominated by entropic changes (delta S++). At equilibrium, rhodamine phalloidin binding generates a positive entropy change (delta S0). The rates of rhodamine phalloidin binding are independent of the pH, ionic strength, and filament length. Rhodamine covalently bound decreases the association rate and affinity of phalloidin for actin. The association rate constant is low for both phalloidin and rhodamine phalloidin because the filaments must undergo conformational changes (i.e. "breathe") to expose the phalloidin binding site [De La Cruz, E. M., & Pollard, T. D. (1994) Biochemistry 33, 14387-14392]. Raising the solvent microviscosity, but not the macroviscosity, dampens these conformational fluctuations, and phalloidin binding kinetics are inhibited. Yeast actin filaments bind rhodamine phalloidin more rapidly, suggesting that perhaps they are more flexible and can breathe more easily than rabbit or Acanthamoeba actin filaments.
我们在22摄氏度下，于含有50 mM氯化钾、1 mM氯化镁且pH值为7.0的缓冲液中，比较了若丹明鬼笔环肽与从兔骨骼肌、卡氏棘阿米巴和酿酒酵母中纯化得到的肌动蛋白结合的动力学和热力学。酿酒酵母肌动蛋白丝与若丹明鬼笔环肽的结合比棘阿米巴和兔骨骼肌肌动蛋白丝更弱，尽管其结合速率常数明显更快，但解离速率更快。与兔骨骼肌肌动蛋白丝相比，若丹明鬼笔环肽对酿酒酵母肌动蛋白丝的解离速率常数更高且结合亲和力更低，这为酵母肌动蛋白丝染色效率低下提供了定量解释[克朗等人（1992年），《美国国家科学院院刊》89卷，4466 - 4470页]。根据过渡态理论解释了速率常数的温度依赖性。基态与过渡态之间存在较小的焓差（ΔH++）。因此，若丹明鬼笔环肽结合和解离的活化自由能（ΔG++）主要由熵变（ΔS++）决定。在平衡状态下，若丹明鬼笔环肽结合会产生正的熵变（ΔS0）。若丹明鬼笔环肽的结合速率与pH值、离子强度和丝长度无关。共价结合的若丹明会降低鬼笔环肽与肌动蛋白的结合速率和亲和力。鬼笔环肽和若丹明鬼笔环肽的结合速率常数都很低，因为肌动蛋白丝必须经历构象变化（即“呼吸”）以暴露鬼笔环肽结合位点[德拉·克鲁兹，E.M.，& 波拉德，T.D.（1994年），《生物化学》33卷，14387 - 14392页]。提高溶剂的微观粘度而非宏观粘度会抑制这些构象波动，进而抑制鬼笔环肽的结合动力学。酵母肌动蛋白丝与若丹明鬼笔环肽的结合更快，这表明它们可能比兔或棘阿米巴肌动蛋白丝更具柔韧性，能更轻松地“呼吸”。
