Reichert A, Heintz D, Echner H, Voelter W, Faulstich H
Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
FEBS Lett. 1996 Jun 3;387(2-3):132-6. doi: 10.1016/0014-5793(96)00488-7.
We have recently described a method for identifying contact sites between actin and thymosin beta4 (Tbeta4) by following spectrophotometrically the extent and kinetics of distinct, thiol-specific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et a]. (1996) J. Biol. Chem. 271, 1301-1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin-DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the KD value of Tbeta4 from the actin-DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tbeta4. In conclusion, DNase I and Tbeta4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin beta4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tbeta4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tbeta4 and actin by X-ray analysis.
我们最近描述了一种通过分光光度法跟踪两种蛋白质的适当衍生物之间独特的、硫醇特异性交联反应的程度和动力学来鉴定肌动蛋白与胸腺素β4（Tβ4）之间接触位点的方法[赖歇特等人（1996年）《生物化学杂志》271卷，1301 - 1308页]。在本研究中，该方法用于表明这种指示复合物形成的交联反应，在肌动蛋白 - DNase I复合物中与在纯肌动蛋白中发生的程度相同，尽管速率稍低。凝胶电泳为三元复合物的形成提供了进一步的证据。通过荧光光谱法发现，肌动蛋白 - DNase I复合物中Tβ4的KD值与纯肌动蛋白中的相同。与这些数据一致，添加Tβ4并不影响肌动蛋白抑制DNase I的能力。总之，DNase I和Tβ4在与肌动蛋白的相互作用中相互独立，这表明胸腺素β4和DNase I在肌动蛋白上的结合位点不重叠。因此，如果以晶体形式获得DNase I、肌动蛋白和Tβ4的三元复合物，就可以为通过X射线分析研究Tβ4与肌动蛋白的界面提供一种方法。
