Binding of cytochalasin D to platelet and muscle myosin.

Human blood platelets exposed to 2 UM tritiated cytochalasin D showed maximum incorporation within 5 min. Tritiated cytochalasin D was found to be bound to thrombosthenin M (platelet myosin), when subjected to gel exclusion chromatography in 0.6 M KCl. Using a Millipore filtration technique, binding at low ionic strength of tritiated cytochalasin D to purified preparations of contractile proteins, showed that platelet thrombosthenin M and muscle myosin, but not actin, was involved (0.9 mole of cytochalasin D per mole of protein). Prior treatment of these proteins with nonradioactive cytochalasin D, followed by exposure to the labeled alkaloid, showed little exchange under these conditions. Binding data gave a value of 0.99 f 0.11 sites per 4.6 X 10” g of muscle myosin, an average intrinsic association constant of 6.2 X lo7 M-I with an heterogeneity index of 1. Although the Ca2+or Mg2+-stimulated ATPase activities of actomyosin from platelets (thrombosthenin) or muscle were not inhibited by 2 UM cytochalsin D, this concentration depressed thrombosthenin M and myosin ATPase. Recombination experiments indicated that actin and cytochalasin D compete for a binding region on myosin. The inhibitory effect of cytochalasin D on myosin ATPase was restored following dialysis against 0.6 M KCl; with concomitant removal of the drug. Furthermore, quantitative measurements of superprecipitation indicated that, although cytochalasin D did not interfere with dissociation of actomyosin by MgATP, increasing concentrations of cytochalasin D inhibited reassociation. Labeled cytochalasin D associated with thrombosthenin M could be removed by solvent extraction on thin layer chromatography and by sodium dodecyl sulfate gel electrophoresis, suggesting that the bond between cytochalasin D and thrombosthenin M or muscle myosin is not covalent.