The accessibility of the thiol groups on G- and F-actin of rabbit muscle.
The accessibility of the cysteine residues of actin from rabbit muscles to the thiol-targeted reagent 7-dimethylamino-4-methyl-(N-maleimidyl)coumarin (DACM) was investigated. Under conditions where the actin is in the unpolymerized form (G-actin), the most reactive thiol group was Cys-257, suggesting that it was located on the surface of the actin molecule. The selective modification of Cys-374 for this reagent as reported by Sutoh [(1982) Biochemistry 21, 3654-3661] was not observed. Cys-10, Cys-217 and Cys-374 were much less reactive and only gradually became extensively modified when the concentration of DACM approached 5 molar equivalents of actin. Presumably these thiol groups were located further inward away from the surface or situated in a different environment that rendered them less reactive. On the other hand, Cys-285 was completely inaccessible and presumably was buried. The lack of preferential labelling of Cys-374 by DACM is incompatible with the finding with iodoacetic acid as the reagent as reported by Elzinga & Collins [(1975) J. Biol. Chem. 250, 5897-5905]. This discrepancy, however, might well be due to the different reagents employed. The DACM-G-actin largely retained its competence for polymerization. Upon polymerization of G-actin, practically all the thiol groups became inaccessible to DACM, suggesting that a drastic change occurred in the conformation of actin units in the transition of monomers to filamentous actin.
Liu
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DF
Department of Biology, Peking University, Beijing, China.
Wang
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D
Stracher
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D000199:Actins / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine; D008301:Maleimides; D009132:Muscles; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013439:Sulfhydryl Reagents
D000199:Actins; D008301:Maleimides; D013439:Sulfhydryl Reagents; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D000255:Adenosine Triphosphate; D003545:Cysteine