INTERACTIONS OF ACANTHAMOEBA-PROFILIN WITH ACTIN AND NUCLEOTIDES BOUND TO ACTIN

Three methods, fluorescence anisotropy of rhodamine-labeled profilin, intrinsic fluorescence and nucleotide exchange, give the same affinity , K-d = 0.1 mu M, for Acanthamoeba profilins binding amoeba actin mono mers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinit y of profilin for actin, The affinity for rabbit skeletal muscle actin is about 4-fold lower. The affinity for both actins is 5-8-fold lower with ADP bound to actin rather than ATP. Pyrenyliodoacetamide labelin g of cysteine 374 of muscle actin reduces the affinity for profilin 10 -fold. The affinity of profilin for nucleotide-free actin is similar t o 3-fold higher than for Mg-ATP-actin and similar to 24-fold higher th an for Mg-ADP-actin. As a result, profilin binding reduces the affinit y of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. ME-ATP dissociate s 8 times faster from actin-profilin than from actin and binds actin-p rofilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as f ast as actin. Thus, profilin promotes the exchange of ADP for ATP. The se properties allow profilin to bind a high proportion of unpolymerize d ATP-actin in the cell, suppressing spontaneous nucleation but allowi ng free barbed ends to elongate at more than 500 subunits/second.