Isolation of the phosphatidylinositol 4-monophosphate dissociable high-affinity profilin-actin complex.
Profilin was originally discovered in a tight complex with monomeric actin from bovine spleen, leading to its description as an actin monomer sequestering protein that maintains a pool of unpolymerized actin in cells. Subsequent purifications of profilin using different methods from diverse cells have consistently yielded preparations that affect the kinetics of actin assembly but do not efficiently maintain actin monomeric at steady state in solutions containing mM magnesium. Recent evidence that profilin inhibits phospholipase C and enhances nucleotide exchange of actin has led some to question whether profilin is ever truly an actin monomer sequestering agent. Here we report that the extraction of bovine spleen with fluoride- and pyrophosphate-containing solutions facilitates isolation of monomeric actin that is bound to profilin and does not polymerize in mM magnesium ion. The integrity of this complex depends on the presence of ATP. Phosphatidylinositol 4-monophosphate (PIP), previously shown to dissociate the low-affinity profilin-actin complex (Kd = 0.4 microM in mM Mg2+), also dissociates the high-affinity profilin-actin complex (Kd less than 0.02 microM in mM Mg2+) yielding actin that is polymerization competent and profilin that functions like profilins purified by conventional methods. Although the chemical basis of these results is not known, they indicate that profilin can tightly sequester actin monomers and support the earlier suggestion that the affinity of profilin for actin may be under metabolic control.
Katakami
Y
Y
Department of Medicine, Brigham and Women's Hospital, Boston, MA.
Katakami
N
N
Janmey
P A
PA
Hartwig
J H
JH
Stossel
T P
TP
D000199:Actins / Q000737:chemistry*; D000255:Adenosine Triphosphate; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003285:Contractile Proteins; D007525:Isoelectric Focusing; D008274:Magnesium; D008840:Microfilament Proteins / Q000737:chemistry*; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D011189:Potassium Chloride; D051304:Profilins; D013154:Spleen / Q000737:chemistry
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D051304:Profilins; C037178:phosphatidylinositol 4-phosphate; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D008274:Magnesium