Drewes G, Faulstich H
Max-Planck-Institut für Medizinische Forschung, Heidelberg, Federal Republic of Germany.
J Biol Chem. 1990 Feb 25;265(6):3017-21.
[Cys374]glutathionyl-actin was prepared by isolation of the reaction product of G-actin with Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid], followed by reaction with glutathione. Filaments of this actin disulfide are susceptible to even weak shearing stress as exerted, for example, by heating to 37 degrees C. This treatment produces a 25-fold enhanced steady-state ATPase activity as compared to unsubstituted F-actin at room temperature. Monitoring the reduction of this enhanced ATPase activity is a reliable method for quantifying the effectiveness of filament-stabilizing agents and for determining their apparent dissociation constants. A detailed comparative study of filament-stabilizing agents was performed, and some hitherto unknown filament-protecting effects were revealed. Inorganic phosphate provides stabilization only to a maximum of 45% ATPase inhibition, but reaches this effect already at cytoplasmic Pi concentrations (approximately 4 mM). Arsenate seems to bind with similar affinity, but with distinctly less protective activity (maximum of 16%). High concentrations of alkali ions provide a more effective protection (maximum of 95%), Li+ being more efficient than Na+ and K+. Divalent cations (Ca2+, Mg2+) had a strong stabilizing effect on KCl-polymerized actin; we confirmed the presence of two distinct classes of binding sites for divalent metal ions with moderate and low affinities, apparent in a strong stabilizing effect on KCl-polymerized actin. The stabilizing effects of KCl and Pi are independent and additive. Correspondingly, at K2HPO4 concentrations greater than 4 mM, K+ ions contribute considerably to stabilization. In the presence of 100 mM KCl plus 4 mM Pi, conditions which mimic the physiological environment, filament protection is nearly as effective as with the mushroom toxin phalloidin. The strong stabilizing effect of phalloidin occurred at concentrations far below stoichiometric, suggesting a very high degree of cooperativity in its interaction with actin filaments.
通过分离G-肌动蛋白与埃尔曼试剂（5,5'-二硫代双（2-硝基苯甲酸））的反应产物，然后与谷胱甘肽反应，制备了[Cys374]谷胱甘肽基-肌动蛋白。这种肌动蛋白二硫化物丝甚至对例如加热至37℃所施加的微弱剪切应力敏感。与室温下未取代的F-肌动蛋白相比，这种处理使稳态ATP酶活性提高了25倍。监测这种增强的ATP酶活性的降低是定量细丝稳定剂有效性和确定其表观解离常数的可靠方法。对细丝稳定剂进行了详细的比较研究，揭示了一些迄今未知的细丝保护作用。无机磷酸盐仅能提供最大45%的ATP酶抑制作用的稳定效果，但在细胞质Pi浓度（约4 mM）时就已达到这种效果。砷酸盐似乎具有相似的亲和力，但保护活性明显较低（最大16%）。高浓度的碱金属离子提供了更有效的保护（最大95%），Li+比Na+和K+更有效。二价阳离子（Ca2+、Mg2+）对KCl聚合的肌动蛋白有很强的稳定作用；我们证实了存在两类具有中等和低亲和力的二价金属离子结合位点，这在对KCl聚合的肌动蛋白的强稳定作用中很明显。KCl和Pi的稳定作用是独立且相加的。相应地，在K2HPO4浓度大于4 mM时，K+离子对稳定作用有很大贡献。在模拟生理环境的100 mM KCl加4 mM Pi的条件下，细丝保护几乎与用蘑菇毒素鬼笔环肽一样有效。鬼笔环肽的强稳定作用在远低于化学计量浓度时就出现了，这表明其与肌动蛋白丝相互作用时具有非常高的协同性。
