Nucleotide in monomeric actin regulates the reactivity of the thiol groups.
A new thiol reagent, 2,4-dinitrophenyl glutathionyl disulfide, allowed the characterization of four thiol groups in monomeric actin by stoichiometric reaction. The number of thiol groups exposed to the reagent was found to depend on the nucleotide bound. In the absence of ATP, G-actin exposed four thiol groups ( G4s ). On the addition of ATP (1 equiv), three of them were shielded. The resulting actin with one thiol group exposed ( G1s ) is the form of monomeric actin normally produced by depolymerization of F-actin in buffers containing ATP. G1s is stable over hours, while G4s , i.e., monomeric actin in ATP-free solution, is not. This must be concluded from the fact that the shielding effect of thiol groups induced by addition of ATP was lost within ca. 30 min probably due to denaturation of G4s to G4s *. Therefore, denaturation of monomeric actin must be understood in terms of loss of thiol shielding, rather than by oxidation of the thiol groups. Addition of equimolar amounts of Ca2+ significantly retarded the denaturation process. ADP (50 equiv) shielded only ca. two of the four thiol groups but, similar to ATP, protected actin from denaturation. Three ATP analogues (10 equiv) were tested but had no shielding effect. In the presence of these analogues actin ( G4s ) rapidly denatured (to G4s *) as in the absence of added nucleotides. It was shown that the thiol-shielding activity and the protective capacity of a nucleotide are interrelated with its binding capability to monomeric actin. G1s was found to be polymerizable as was G approximately 2s on the addition of ATP. No polymerization could be detected for G4s or G4s *.(ABSTRACT TRUNCATED AT 250 WORDS)
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D009132:Muscles / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
C032599:2,4-dinitrophenyl-glutathionyl-disulfide; D000199:Actins; D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D005978:Glutathione