Interaction of G-actin with thymosin beta 4 and its variants thymosin beta 9 and thymosin beta met9.
Thymosin beta 4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin beta 9 and thymosin beta met9 are minor variants of thymosin beta 4. The possible function of these peptides has been investigated by comparing the actin binding properties of these beta-thymosins. Thymosin beta 9 and thymosin beta met9 were found to inhibit polymerization of ATP-actin with identical KDs of 0.7-0.8 microM (as compared to 2 +/- 0.3 microM 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 met9 with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin beta 4 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 beta 4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of approximately 1.5 microM-1 s-1 and 2 s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin beta 4 function in vivo is discussed.
Jean
C
C
Laboratoire d'Enzymologie, Gif-sur-Yvette, France.
Rieger
K
K
Blanchoin
L
L
Carlier
M F
MF
Lenfant
M
M
Pantaloni
D
D
D000199:Actins / Q000032:analysis / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D004347:Drug Interactions; D005453:Fluorescence; D014644:Genetic Variation; D008433:Mathematics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011108:Polymers; D011485:Protein Binding; D011817:Rabbits; D013947:Thymosin / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000099201:Thymosin beta(4)
D000199:Actins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008715:Methionine; D011108:Polymers; D013947:Thymosin; D000099201:Thymosin beta(4); C067214:thymosin beta(9), methionine; C042362:thymosin beta(9)