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, Kd = 0.1 microM, for Acanthamoeba profilins binding amoeba actin monomers with bound Mg-ATP. Replacement of serine 38 with cysteine created a unique site where labeling with rhodamine did not alter the affinity 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 labeling of cysteine 374 of muscle actin reduces the affinity for profilin 10-fold. The affinity of profilin for nucleotide-free actin is approximately 3-fold higher than for Mg-ATP-actin and approximately 24-fold higher than for Mg-ADP-actin. As a result, profilin binding reduces the affinity of actin 3-fold for Mg-ATP and 24-fold for Mg-ADP. Mg-ATP dissociates 8 times faster from actin-profilin than from actin and binds actin-profilin 3 times faster than actin. Mg-ADP dissociates 14 times faster from actin-profilin than from actin and binds actin-profilin half as fast as actin. Thus, profilin promotes the exchange of ADP for ATP. These properties allow profilin to bind a high proportion of unpolymerized ATP-actin in the cell, suppressing spontaneous nucleation but allowing free barbed ends to elongate at more than 500 subunits/second.
Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
V K
VK
Vinson
E M
EM
De La Cruz
H N
HN
Higgs
T D
TD
Pollard
D000048:Acanthamoeba / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D003285:Contractile Proteins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D007211:Indoles / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000199:Actins; D003285:Contractile Proteins; D005456:Fluorescent Dyes; D007211:Indoles; D008840:Microfilament Proteins; D051304:Profilins; D015800:Protozoan Proteins; D012235:Rhodamines; C082337:rim 1; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate