Direct extraction of G-actin from the myosin-removed myofibrils under the conditions of low ionic strength.
Muscle actin is, in most cases, prepared from an acetone-dried powder of the myosin-removed myofibrils under low-salt conditions in the presence of ATP. In this paper, it is shown that G-actin can be directly extracted from the myosin-removed myofibrils without acetone treatment. The extraction conditions are the same as those used for the extraction of G-actin from the dried powder: extraction of the myosin-removed myofibrils for 1 h with 2 mM Tris-HCl, pH 8.0, in the presence of 0.5 mM ATP. However, the crude G-actin directly extracted from the myosin-removed myofibrils loses its polymerizability after prolonged extraction. Measurements of inorganic phosphate and thin layer chromatography of the adenine nucleotides of the crude G-actin solution show that free ATP added to the extraction buffer is sequentially hydrolyzed to ADP and AMP, and then finally converted to IMP. The instability of the G-(ADP)-actin, depolymerized from the ends of actin filaments, explains the loss in polymerizability of G-actin during the extraction. Residual ATPase, adenylate kinase, and deaminase contained in the myofibrils may account for the decomposition of ATP.
D000199:Actins / Q000302:isolation & purification*; D000227:Adenine Nucleotides / Q000032:analysis; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002645:Chickens; D002855:Chromatography, Thin Layer; D009210:Myofibrils / Q000032:analysis*; D009218:Myosins / Q000032:analysis; D009994:Osmolar Concentration; D011189:Potassium Chloride; D013997:Time Factors
D000199:Actins; D000227:Adenine Nucleotides; D011189:Potassium Chloride; D009218:Myosins