Thiol group reactivity and polymerization of actin in the presence of ATP analogs.
We have investigated polymerization and the number of SH-groups of monomeric actin exposed in the presence of (beta, gamma)-substituted ATP-analogs. Actin, when depolymerized in a buffer containing 10 equiv. of APPCP exposes 4 thiol groups. The time course of the SH-titration is similar to that obtained when F-actin is depolymerized in a nucleotide free buffer. When actin is depolymerized in a buffer containing 10 equiv. of APPNP it also exposes 4 thiols. However, thiol-titration follows different kinetics. While one SH group reacts quickly the reaction of 3 others is retarded. We conclude that APPNP exhibits a shielding effect on part of the thiols for a period of time, while APPCP does not. In agreement with this, in the presence of APPNP yield of polymerization as well as stability against denaturation are distinctly higher than without added nucleotide or in the presence of APPCP. In line with this a hydrolysis product, most probably APPNH2, was associated with the filaments, as indicated by the replacement of tritiated ADP during polymerization, and from analysis of the attached nucleotide. Under the same conditions APPCP replaced tritiated ADP only to a small extent. The data indicate that APPNP interacts with monomeric actin much less than ATP and still less than ADP, but more so APPCP. APPNP is cleaved by actin ATPase and a hydrolysis product is incorporated into filaments.
Department of Basic Sciences, University of Crete, Iraklion, Greece.
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Stournaras
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Merkler
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Faulstich
D000199:Actins / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D007700:Kinetics; D011108:Polymers; D011489:Protein Denaturation; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis*
D000199:Actins; D011108:Polymers; D013438:Sulfhydryl Compounds; D000266:Adenylyl Imidodiphosphate; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate