Preparation and polymerization of skeletal muscle ADP-actin.
Skeletal muscle ADP-G-actin was prepared from ADP-F-actin, which had been freed of residual ATP by repeated sonication, by depolymerization in 5 mM Tris-HCl, 0.2 mM ADP, 0.2 mM dithiothreitol, 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.01% NaN3, pH 8.0. The ADP had been freed of traces of ATP by DEAE-chromatography, and 5 microM diadenosine pentaphosphate was added to inhibit myokinase activity. The kinetics of the spontaneous polymerization of ADP-actin in 1 mM MgCl2 + 0.1 M KCl were compatible with the simple nucleation-elongation model previously used to explain the polymerization of ATP-actin. The critical concentrations of ADP-actin were 8.0 and 2.0 microM in 1 mM MgCl2 and 1 mM MgCl2 + 0.1 M KCl, respectively. These values are 20-30-fold higher than the corresponding values in ATP. Using cross-linked actin trimers to nucleate polymerization, the association rate constants were found to be 0.8 and 0.9 microM-1 S-1 in MgCl2 and MgCl2 + KCl, respectively, which are 0.4 and 0.2 times the values for ATP-actin. The dissociation rate constants, calculated from the critical concentrations and the association rate constants, were 6.4 and 1.8 S-1, respectively, which are 10 and 5 times the corresponding values for ATP-actin.
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D009132:Muscles / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits
C042634:ADP-G-actin; D000199:Actins; D046911:Macromolecular Substances; D015636:Magnesium Chloride; D000244:Adenosine Diphosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D008274:Magnesium