Purification and characterization of an ATP-sensitive actin gelation protein from Acanthamoeba castellanii.
A protein which cross-links actin filaments in a nucleotide-sensitive manner has been purified to homogeneity from Acanthamoeba castellanii. This protein, GF-210, is a slightly asymmetric molecule composed of six subunits, each with an apparent mass of 35,000 Da. As determined by the method of falling ball vicometry, GF-210 was shown to cross-link actin filaments at hexamer:actin molar ratios of 1:500, with gelation occurring at molar ratios of 1:300 and higher. Actin gels did not form in the presence of 10 microM ATP, and filament cross-linking was completely inhibited by 100 microM ATP. Although ATP was the most effective inhibitor of actin filament cross-linking, other phospho-compounds including ADP, GTP, sodium phosphate, and sodium pyrophosphate prevented gelation at concentrations lower than 1.5 mM. In contrast, 50 mM KCl was required to inhibit the formation of actin networks. Direct binding studies showed that GF-210 binds to F-actin with a KD of 1.2 microM in the absence of ATP but with a KD of 72.8 microM in the presence of 2 mM ATP. This weakening of the interaction between F-actin and GF-210 may explain the inhibition of GF-210-induced actin cross-linking by nucleotides and other phospho-compounds.
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000656:Amoeba / Q000032:analysis*; D000818:Animals; D002352:Carrier Proteins; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011189:Potassium Chloride / Q000494:pharmacology
D000199:Actins; D002352:Carrier Proteins; C034037:F-actin-binding proteins; D005782:Gels; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D011189:Potassium Chloride; D000255:Adenosine Triphosphate