Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin.
We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.
Kinosian
Henry J
HJ
Center for Cell Biology and Cancer Research, Department of Medicine, Albany Medical College, Albany, NY 12208. USA. henry.kinosian@med.va.gov
Selden
Lynn A
LA
Gershman
Lewis C
LC
Estes
James E
JE
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003285:Contractile Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry; D011108:Polymers / Q000378:metabolism; D051304:Profilins; D011817:Rabbits; D013154:Spleen / Q000737:chemistry; D013816:Thermodynamics
D000199:Actins; D003285:Contractile Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D011108:Polymers; D051304:Profilins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate