Interaction of Actin and Myosin in the Absence and Presence of ATP

Abstract

The binding between F-actin and myosin has been studied by analyzing the amount of radio-actively labelled (by means of 14C-NEM) F-actin and myosin in the formed unsoluble actomyosin pellet after centrifugation of the reaction mixture. In the absence of ATP, the amount of actin bound to myosin varies, depending on the amounts of actin and myosin present, between 0.18 mg actin/mg myosin (2 actin units per 1 myosin molecule) and about 2 mg actin/mg myosin (each actin fibril only uncompletely saturated with myosin). In 0.03 ᴍ KCl ATP decreases, if at all, the affinity of actin towards myosin only slightly; but less actin is bound to myosin in the presence of MgATP and in low ionic strength, indicating that myosin is now more densily distributed over fewer actin fibrils leaving the rest of fibrils free. For precipitation of the total amount of myosin (myosin alone is soluble in MgATP) incomplete saturation of myosin with actin suffices; obviously actin promotes filament formation of myosin. The activation of myosin ATPase by actin depends in a similar manner as actin binding does on actin concentration, hence the enzymatic interaction between actin and myosin is accompanied by true actin-myosin binding. An actin-tropomyosin-troponin preparation, whose reduced viscosity is lower than that of F-actin and which consists of about 30% actin, activates myosin ATPase to the same extent as F-actin does. It competes with F-actin for the same binding sites on myosin and can in the presence of MgATP be displaced from myosin by those preparations of F-actin which have a strong tendency to become fully saturated with myosin. The activation of myosin ATPase by actin-tropomyosin-troponin is reduced after tryptic digestion of actin-tropomyosin-troponin, which affects, according to SDS gel electro­ phoresis, mainly two components of troponin with molecular weights of about 32 000 and 25 000, respectively

Changes in the State of Actin during Superprecipitation of Actomyosin

“…Partial solubilization of actin in actomyosin suspensions at low ionic strength in the presence of millimolar concentrations of MgATP reported in this work has been recently observed also by Dancker and Hoffmann [41] with the use of actin labelled with radioactive N-ethylmaleimide. In both cases the release of actin was observed even if total actin content in actomyosin was far below the saturating ratio of two actin monomers per one myosin molecule (about one part of actin per five parts of myosin, weight ratio) deduced from maximum activation of myosin ATPase [42] and ultracentrifugal studies on binding of H-meromyosin to actin in the absence of ATP [43,44].…”

“…It has been shown by Dancker and Hoffmann [41] and indicated by the results obtained in this work that, contrary to ATP, inorganic pyrophosphate in the presence of Mg2+ does not appreciably change the binding between actin and myosin. Evidently, the known dissociating effect of this reagent is easily manifested only at high ionic strength, consistently with the observation that the actomyosin system of glycerinated muscle fibres is much less sensitive to it (as judged from tension measurements) than the dissolved actomyosin (as indicated by viscosity measurements) [46].…”

Dissociation of actomyosin complex by monovalent fragments of polyclonal antibodies to myosin

“…In incubates of actomyosin with specific Fab, three components were noted: a minor one sedimenting with an ~20 of 18.2 (non-dissociated actomyosin), a major one with an ~20 of 9 (myosin-anti-myosin Fab complexes, as identified by sedimenting myosin-anti- The experiments of Dancker and Hoffmann [3] have suggested a stability constant for the actomyosin complex of about 2 x LO6 M-' and thus a high affinity of actin to myosin. Our above data give evidence that the affinity of the myosin antibodieseven in their less functional monovalent state -must be even higher in order to dissociate the complex.…”

Spectroscopic evidence for the interaction of phalloidin with actin

“…Bovine serum albumin was a product of Behringwerke, Marburg. F-actin, free from troponin and tropomyosin was a gift of P. Dancker and M. Hoffmann, who prepared it from rabbit sketetal muscle according to [4] . The F-actin pellets were homogenized in 0.1 M KC1 to a final concentration of 1.48 X IO-' M. Concentrations of actin and phalloidin were determined spectrophotometrically.…”