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Abstract

We have recently described a method for identifying contact sites between actin and thymosin β4 (Tβ4) by following spectrophotometrically the extent and kinetics of distinct, thiolspecific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et al. (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 K D value of Tβ4 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 Tβ4. In conclusion, DNase I and Tβ4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin β4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tβ4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tβ4 and actin by X-ray analysis.