Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments.
I measured the rate of elongation at the barbed and pointed ends of actin filaments by electron microscopy with Limulus sperm acrosomal processes as nuclei. With improvements in the mechanics of the assay, it was possible to measure growth rates from 0.05 to 280 s-1. At 22 degrees C in 1 mM MgCl2, 10 mM imidazole (pH 7), 0.2 mM ATP with 1 mM EGTA or 50 microM CaCl2 or with EGTA and 50 mM KCl, the elongation rates at both ends have a linear dependence on the ATP-actin concentration from the critical concentration to 20 microM. Consequently, over a wide range of subunit addition rates, the rate constants for association and dissociation of ATP-actin are constant. This shows that the nucleotide composition at or near the end of the growing filament is either the same over this range of growth rates or has no detectable effect on the rate constants. Under conditions where polymerization is fastest (MgCl2 + KCl + EGTA) the rate constants have these values: (table; see text) Compared with ATP-actin, ADP-actin associates slower at both ends, dissociates faster from the barbed end, but dissociates slower from the pointed end. Taking into account the events at both ends, these constants and a simple Oosawa-type model account for the complex three-phase dependence of the rate of polymerization in bulk samples on the concentration of ATP-actin monomers observed by Carlier, M.-F., D. Pantaloni, and E. D. Korn (1985, J. Biol. Chem., 260:6565-6571). These constants can also be used to predict the reactions at steady state in ATP. There will be slow subunit flux from the barbed end to the pointed end. There will also be minor fluctuations in length at the barbed end due to occasional rapid dissociation of strings of ADP subunits but the pointed end will be relatively stable.
D000177:Acrosome / Q000648:ultrastructure; D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008854:Microscopy, Electron; D008871:Microvilli / Q000648:ultrastructure; D011817:Rabbits
D000199:Actins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate