THE END OF A POLYMERIZING ACTIN FILAMENT CONTAINS NUMEROUS ATP-SUBUNIT SEGMENTS THAT ARE DISCONNECTED BY ADP-SUBUNITS RESULTING FROM ATP HYDROLYSIS

ATP hydrolysis by copolymers of ATP-actin and ADP-actin was investigat ed in order to analyze the effect of interfaces between ATP-subunits a nd ADP-subunits on hydrolysis of actin-bound ATP. Copolymers of ATP- a nd ADP-subunits were formed by polymerization of ATP- and ADP-actin mo nomers onto filaments. By changing the ratio of polymerizing ATP-actin monomers to ADP-actin monomers, the number of interfaces between ATP- and ADP-subunits and of ATP-subunits only surrounded by further ATP-s ubunits was varied. The rate of actin polymerization and of ATP hydrol ysis was measured simultaneously on the same samples. The lag time bet ween incorporation of actin monomers into filaments and subsequent ATP hydrolysis was found to be similar both for polymerized ATP-actin and for copolymers formed by various ratios of ATP- to ADP-actin. The exp eriments were performed in the presence of 1 mM MgCl2, 0.05 mM CaCl2, and 100 mM KCl or of 1 mM MgCl2 and 0.4 mM EGTA. The type of cations w as found to have no major effect on the rate of ATP hydrolysis. A quan titative evaluation of the experimental data suggests that ATP at inte rfaces between ATP- and ADP-subunits is hydrolyzed not more than 10 ti mes faster than ATP of subunits surrounded by further ATP-subunits. On the basis of these results, one can conclude that an actin filament o nto which ATP-actin monomers polymerize contains numerous segments of ATP-subunits that are disconnected by ADP-subunits resulting from ATP hydrolysis. The average length of the numerous ATP segments of a stead ily polymerizing filament is in the range of Ip ATP-subunits or below.