Interaction of G-actin with thymosin ?4 and its variants thymosin ?9 and thymosin ? 9 met

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Abstract
Thymosin β4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin β9 and thymosin β 9 met are minor variants of thymosin β4. The possible function of these peptides has been investigated by comparing the actin binding properties of these β-thymosins. Thymosin β9 and thymosin β 9 met were found to inhibit polymerization of ATP-actin with identical K d s of 0.7–0.8 μM (as compared to 2±0.3 μM for thymosin β4); like thymosin β4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin β4 and thymosin β 9 met with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin β4 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin β4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of ∼1.5 μM-1 s-1 and 2s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin β4 function in vivo is discussed.