Chemical evidence for the existence of activated G-actin.
Globular actin (G-actin) will polymerize to form filamentous actin (F-actin) under physiological ionic conditions, and is known to be regulated by univalent and bivalent cations, such as K+ and Mg2+. The current concept of this process involves four steps: activation, nucleation, elongation and annealing. Evidence for the existence of activated G-protein has been suggested by changes in the resistance to proteolysis [Rich & Estes (1976) J. Mol. Biol. 104, 777-792] and u.v.-light absorption [Rouayrenc & Travers (1981) Eur. J. Biochem. 116, 73-77]. More recently we [Liu et al. (1990) Biochem. J. 266, 453-459] have provided direct chemical evidence for extensive conformational changes during the transformation of G-actin into F-actin. In this study we now present direct chemical evidence for the existence of a short-lived species, an activated form of G-actin, which can be detected by changes in the accessibility of the free thiol groups on the G-actin molecule when modified by a specific thiol-group-targeted reagent, 7-dimethylamino-4-methyl-3-N-maleimidylcoumarin (DACM). The presence of K+ and/or Mg2+ ions caused a large increase in the accessibility of the thiol groups of Cys-217 and Cys-374, but not those of Cys-10 and Cys-257. Mg2+ effected relatively faster changes than did K+ ions. The results suggest that the function of these ions is to convert G-actin into an activated form, and further suggest that the change in conformation is mainly confined to the large domain. Such changes at least involve certain portions of the G-actin molecule that contain Cys-217 and Cys-374. On the other hand, little or no significant change could be observed in the small domain of G-actin as reflected by the accessibility of Cys-10. The bound nucleotide remained as ATP during the activation of G-actin and was hydrolysed to ADP on polymerization. The activated G-actin had a life-time of about 8 min or less depending on the concentration of G-actin. At higher protein concentration, its life-time was much shorter, probably owing to the earlier onset of polymerization, which apparently is governed by the concentration of the activated form. The life-time of this new species can be extended by lowering the temperature and is less affected by actin concentration. This new species is considered to be an activated form of G-actin, since polymerization renders all the thiol groups on actin inaccessible to the reagent DACM.
Shu
W P
WP
Department of Biochemistry, SUNY Health Science Center, Brooklyn 11203.
Wang
D
D
Stracher
A
A
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D003488:Cyanogen Bromide; D007525:Isoelectric Focusing; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009132:Muscles / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000199:Actins; D008301:Maleimides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D008274:Magnesium; D003488:Cyanogen Bromide; D011188:Potassium