Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii.
Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D000656:Amoeba / Q000032:analysis*; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002848:Chromatography, DEAE-Cellulose; D002918:Chymotrypsin / Q000378:metabolism; D051345:Destrin; D004591:Electrophoresis, Polyacrylamide Gel; D018260:Gelsolin; D008840:Microfilament Proteins; D008854:Microscopy, Electron; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011108:Polymers / Q000032:analysis; D011506:Proteins / Q000032:analysis; D015800:Protozoan Proteins; D013215:Starfish; D014783:Viscosity
D051339:Actin Depolymerizing Factors; D000199:Actins; D000596:Amino Acids; D002352:Carrier Proteins; D051345:Destrin; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011108:Polymers; D011506:Proteins; D015800:Protozoan Proteins; C047604:actophorin protein, Acanthamoeba; C016953:brevin; D002918:Chymotrypsin