Document Detail


Chemical evidence for the existence of activated G-actin.
MedLine Citation:
PMID:  1575699     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
W P Shu; D Wang; A Stracher
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Biochemical journal     Volume:  283 ( Pt 2)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1992 Apr 
Date Detail:
Created Date:  1992-06-02     Completed Date:  1992-06-02     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  567-73     Citation Subset:  IM    
Affiliation:
Department of Biochemistry, SUNY Health Science Center, Brooklyn 11203.
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MeSH Terms
Descriptor/Qualifier:
Actins / chemistry,  metabolism*
Animals
Cyanogen Bromide
Isoelectric Focusing
Magnesium / pharmacology
Maleimides / pharmacology
Muscles / metabolism
Peptide Fragments / isolation & purification
Potassium / pharmacology
Protein Conformation
Rabbits
Sulfhydryl Compounds / analysis
Sulfhydryl Reagents / pharmacology
Grant Support
ID/Acronym/Agency:
HL14020/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Actins; 0/Maleimides; 0/Peptide Fragments; 0/Sulfhydryl Compounds; 0/Sulfhydryl Reagents; 506-68-3/Cyanogen Bromide; 55145-14-7/N-(7-dimethylamino-4-methylcoumarinyl)maleimide; 7439-95-4/Magnesium; 7440-09-7/Potassium
Comments/Corrections

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