INTERACTION OF G-ACTIN WITH THYMOSIN BETA(4) AND ITS VARIANTS THYMOSIN BETA(9) AND THYMOSIN BETA(MET)(9)

Thymosin beta(4) is a major actin sequestering peptide in vertebrate c ells and plays a role in the regulation of actin monomer/polymer ratio . Thymosin beta(9) and thymosin beta(9)(met) are minor variants of thy mosin beta(4). The possible function of these peptides has been invest igated by comparing the actin binding properties of these beta-thymosi ns. Thymosin beta(9) and thymosin beta(9)(met) were found to inhibit p olymerization of ATP-actin with identical K(D)s of 0.7-0.8 mu M (as co mpared to 2 +/- 0.3 mu M for thymosin beta(4)); like thymosin beta(4), they bound to ADP-G-actin with a 100-fold lower affinity than to ATP- G-actin. The interaction of thymosin beta(4) and thymosin beta(9)(met) with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin beta(4) to G-actin was acco mpanied by a 15% increase in the fluorescence intensity of actin trypt ophans, and a 10 nm emission blue shift. Methionine-6 played an import ant role in this effect. The fluorescence change was used to monitor t he kinetics of thymosin beta(4) binding to G-actin in the stopped-flow . The reaction was bimolecular, with association and dissociation rate constants of similar to 1.5 mu M(-1) s(-1) and 2 s(-1) respectively, under physiological conditions. The possible physiological significanc es of methionine-6 oxidation and of the relatively slow binding kineti cs in regulating thymosin beta(4) function in vivo is discussed.