Purification and characterization of aginactin, a newly identified agonist-regulated actin-capping protein from Dictyostelium amoebae.
Amoeboid chemotaxis involves a regulated increase in actin nucleation activity that is correlated with an increase in actin polymerization occurring seconds after chemotactic stimulation (Carson, M., Weber, A., and Zigmond, S. H. (1986) J. Cell Biol. 103, 2707-2714; Hall, A. L., Warren, V., Dharmawardhane, S., and Condeelis, J. (1989) J. Cell Biol. 109, 2207-2213). We report the isolation and characterization of an agonist-regulated capping protein, aginactin, from Dictyostelium that may regulate these changes in actin nucleation activity. Aginactin is isolated from low speed supernatants of starved amoebae by sequential anion exchange, hydrophobic interaction, fast protein liquid chromatography anion exchange, and hydroxyapatite chromatography. Aginactin migrates with an apparent molecular weight of 70,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and gel filtration columns, suggesting that it is a globular monomer. Aginactin is a barbed-end capping protein by several criteria. It inhibits the rate and final extent of actin polymerization and increases the apparent critical concentration at substoichiometric ratios to actin. It also inhibits depolymerization of F-actin and inhibits polymerization at the barbed end of Limulus acrosomal bundles. Aginactin is unaffected by micromolar Ca2+, and it neither severs F-actin nor nucleates actin polymerization in either the presence or absence of Ca2+. Aginactin binds to and cosediments with F-actin and has an apparent Kd for capping F-actin of 2.7 nM.
Sauterer
R A
RA
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Eddy
R J
RJ
Hall
A L
AL
Condeelis
J S
JS
D051344:Actin Capping Proteins; D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000818:Animals; D002633:Chemotaxis; D002853:Chromatography, Liquid; D051345:Destrin; D004023:Dictyostelium / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D015800:Protozoan Proteins
D051344:Actin Capping Proteins; D051339:Actin Depolymerizing Factors; D000199:Actins; D051345:Destrin; D005656:Fungal Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; C072098:aginactin protein, Dictyostelium