Interaction of Actomyosin with Adenosine Triphosphate at Low Ionic Strength

Abstract

The clearing of actomyosin at p = 0.15 to 0.16 and pH 7.0 brought about by adenosine triphosphate in the presence of added magnesium was interpreted in the preceding paper in terms of the dissociation of actomyosin into its components (2).This report deals with various factors influencing the clearing reaction and the accompanying changes in adenosine triphosphatase activity.Adenosine triphosphate was found to be most effective among the various nucleoside triphosphates and magnesium to be specific among the divalent metals tested.At p < 0.1, adenosine triphosphate and magnesium alone cannot produce clearing: chelating reagents or the relaxing factor of muscle are necessary.The possible physiological meaning of these findings is briefly discussed. EXPERIMENTAL PROCEDUREThe preparation of myosin and actin was described previously (2).The muscle granules were prepared as follows: rabbit muscle was homogenized in 3 volumes of 0.05 M KHCO,, centrifuged for 20 minutes at 9,000 x g, and the supernatant was centrifuged for 1 hour at 35,000 x g.The precipitated granules were suspended in 0.03 M KHCOI, clarified for 20 minutes at 9,000 X g, and used within 24 hours after the preparation.The increase in the double refraction of flow, measured in an apparatus of the Edsall type, was used as a measure of clearing (2).ATPase activity was determined in most experiments with the use of the pH-Stat system described in the preceding paper; in some experiments the Pi liberation was measured according to Fiske and SubbaRow (3).C?4-ADP-labeled F-actin was prepared according to M.artonosi, Gouvea, and Gergely (4).Free ATP was removed by Dowex 1 treatment (5) instead of prolonged dialysis (4).Samples dried on filter paper strips were counted in a Packard Tri-Carb scintillation counter essentially as described by Loftfield and Eigner (6).