Phosphorylation of Amoeba G-actin and its effect on actin polymerization.
Mass culture of Amoeba proteus enabled us to do biochemical studies on this organism. Actin and profilin were purified from Amoeba to examine actin phosphorylation and polymerization. The apparent molecular weight of Amoeba actin was 44,000, and its isoelectric point was 5.8. The apparent molecular weight of Amoeba profilin was 12,000, and its isoelectric point was 4.9. It reduced the rate of actin polymerization as reported in the cases of profilins from other organisms. A protein of Mr = 44,000 (44 K protein) was phosphorylated in a Ca2+-dependent manner in cell homogenate of Amoeba without being inhibited by calmodulin antagonists. Using the homogenate as a kinase, purified Amoeba G-actin could be phosphorylated in proportion to the amount of actin. However, neither Amoeba F-actin nor rabbit skeletal muscle G-actin was phosphorylated. The phosphorylation of Amoeba actin with a kinase partially purified from A. proteus increased with dilution of the actin concentration. When Amoeba profilin was added, more than 80% of the actin was phosphorylated. By viscometry, electron microscopy, and ultracentrifugation analysis it was demonstrated that Amoeba G-actin phosphorylated in the presence of profilin and kinase did not polymerize in this solution. High-performance liquid chromatography analysis showed that phosphorylated Amoeba actin remained in a monomeric state even under conditions favorable for actin polymerization.
D000199:Actins / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000656:Amoeba / Q000378:metabolism*; D000818:Animals; D003285:Contractile Proteins; D007700:Kinetics; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D008970:Molecular Weight; D010766:Phosphorylation; D051304:Profilins; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism
D000199:Actins; D000596:Amino Acids; D003285:Contractile Proteins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D051304:Profilins; D011506:Proteins