Cytochalasin inhibits the rate of elongation of actin filament fragments

Abstract

Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified Dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B; 5 .times. 10-7 M cytochalasin B lowers the rate to 70% of the control value, whereas 10-7 M cytochalasin D lowers it to 25%. Fragments of F-actin were used to increase the rate of assembly 5-fold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2 .times. 10-7 M and 10-8 M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4 .times. 10-8 M and 1 .times. 10-9 M, respectively. A Scatchard plot has been obtained using [3H]cytochalasin B binding to F-actin in the absence of ATP. The Kd from this plot (.apprx. 4 .times. 10-8 M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly 1/F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.