Interactions of beta-thymosins, thymosin beta 4-sulfoxide, and N-terminally truncated thymosin beta 4 with actin studied by equilibrium centrifugation, chemical cross-linking and viscometry.
All beta-thymosins studied interact with G-actin in a bimolecular complex and inhibit the polymerization to F-actin under high salt conditions. The interactions between actin and beta-thymosins have been studied under polymerization conditions using actin labeled by a fluorescent reporter group at Cys374. Instead of labeling actin we employed equilibrium centrifugation of unlabeled G-actin, viscometry, and chemical cross-linking to investigate the interactions with several beta-thymosins, oxidized thymosin beta 4 and N-terminally truncated beta 4. The apparent dissociation constants for actin from bovine heart and beta-thymosins were 2.5, 0.1, and 2.7 microM for thymosin beta 4, [Ala1]beta 4(beta Ala4), and beta 10, respectively. Comparable apparent dissociation constants were obtained for the interaction of G-actin from rabbit skeletal muscle and thymosin beta 4 or beta Ala4. In rabbits thymosin beta Ala4 replaces beta 4 being different in amino acid residue 1 only. The apparent dissociation constant of thymosin beta 10 with actin from rabbit skeletal muscle, however, is about 10% of the value obtained with actin from bovine heart. Oxidation of thymosin beta 4 at Met6 (beta 4-sulfoxide) as well as truncation of 6 [beta 4-(7-43)] or 12 [beta 4-(13-43)] amino acid residues from the N-terminus increase apparent dissociation constants to 38-53 microM. Truncation of the first 23 amino acid residues [beta 4-(24-43)] abolishes interaction with G-actin completely. Therefore, amino acid residues between position 13 and 24 are necessary for 1-ethyl-3[3-(dimethyl-aminopropyl)-carbodiimide cross-linking of G-actin. In spite of comparable apparent dissociation constants between actin and thymosin beta 4-sulfoxide or beta 4-(7-43) or beta 4-(13-43), only beta 4-sulfoxide and not the truncated beta-thymosins inhibits actin polymerization, however, only at a 20-fold higher concentration than beta 4. Thus the first six amino acid residues are indispensable to inhibit salt-induced actin polymerization as analyzed by viscometry. While the apparent dissociation constant of the actin/thymosin beta 4 complex generated from a preformed actin/DNase-I complex is 160 microM, a fivefold excess of DNase I over the preformed actin/thymosin-beta 4 complex is necessary to observe a comparable dissociation constant.
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Institut für Biochemie, Medizinische Fakultät, Universität Erlangen-Nürnberg, Germany.
Zerzawy
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Hannappel
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D000199:Actins / Q000378:metabolism*; D000818:Animals; D017317:Annexin A6 / Q000235:genetics* / Q000378:metabolism; D001704:Biopolymers; D002417:Cattle; D002498:Centrifugation; D003432:Cross-Linking Reagents; D003850:Deoxyribonuclease I / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D013947:Thymosin / Q000737:chemistry / Q000378:metabolism*; D014783:Viscosity; D000099201:Thymosin beta(4)
D000199:Actins; D017317:Annexin A6; D001704:Biopolymers; D003432:Cross-Linking Reagents; D003850:Deoxyribonuclease I; D008565:Membrane Proteins; D008840:Microfilament Proteins; D013947:Thymosin; D000099201:Thymosin beta(4); C094615:thymosin beta(4) sulfoxide