Isolation and characterization of actin from Entamoeba histolytica.
Actin has been identified and purified partially from trophozoites of Entamoeba histolytica HMI-IMSS by a procedure that minimizes proteolysis. In cellular extracts, Entamoeba actin would copolymerize with muscle actin, but would not bind to DNase I or form microfilaments. Fractionation of the extracts by DEAE-cellulose and Sephadex G-150 chromatography yielded a purified actin that would copolymerize with rabbit skeletal muscle actin or polymerize alone into long filaments at 24 degrees C upon addition of 100 mM KC1 and 2 mM MgCl2. These filaments are not cold-stable and will depolymerize at 4 degrees C in 1 or 2 h. Entamoeba actin filaments bind phallotoxin with the same affinity as muscle actin and decorate with rabbit skeletal muscle heavy meromyosin. Entamoeba actin filaments activate the Mg2+ ATPase of heavy meromyosin to the same Vmax as muscle actin, but the Kapp is 2.8 times higher. Entamoeba actin is a single species with a slightly higher molecular weight than muscle actin (45,000) and a more acidic pI (5.4). The purified actin does not bind to DNase I, produce inhibition of the enzymatic activity, or block the binding of muscle actin. Comparison of the peptides obtained by limit digest with protease V8 from Staphylococcus aureus shows sequences with common mobility between alpha-actin and Entamoeba actin, but additional peptides are present which may account for the different properties of the Entamoeba actin. Finally, in vitro translation of mRNA from trophozoites produces a single polypeptide equivalent to the molecule purified from Entamoeba extracts.
D000199:Actins / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000378:metabolism*; D008854:Microscopy, Electron; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D012156:Reticulocytes / Q000378:metabolism
D000199:Actins; D012333:RNA, Messenger