Formation of a ternary complex: actin, 5'-adenylyl imidodiphosphate, and the subfragments of myosin.
The formation of the ternary complex composed of actin, 5'-adenylyl imidodiphosphate [AMP-P(NH)P], and myosin subfragment 1 (S-1) was studied using the analytical ultracentrifuge with UV optics, which enabled the direct determination of the extent of dissociation of actin.S-1 (acto.S-1) by AMP-P(NH)P. In contrast to the reaction with ATP, at saturating levels of AMP-P(NH)P (1.5 mM), extensive formation of the ternary acto.S-1.AMP-P(NH)P complex occurs at 22 degrees . With 40 muM actin present, AMP-P(NH)P causes almost no dissociation of the acto.S-1 complex at 0.04 M ionic strength, while even at 0.22 M ionic strength one-third of the S-1 remains associated with actin and AMP-P(NH)P in a ternary complex. A detailed study of the binding of S-1.AMP-P(NH)P to actin using the Scatchard plot analysis shows that, at saturation, 1 mol of S-1.AMP-P(NH)P binds per mol of actin monomer. There appears to be no cooperativity occurring as the S-1.AMP-P(NH)P binds along the actin filament, with the possible exception of a slight positive cooperativity when most of the sites on the actin filament are saturated. The turbidity of the ternary complex is identical to the turbidity of acto.S-1 alone. Preliminary experiments with the two-headed subfragment of myosin, heavy meromyosin (HMM), show that the binding of HMM.[AMP-P(NH)P](2) to actin is only about twice as strong as the binding of S-1.AMP-P(NH)P to actin, indicating that the second head contributes very little to the free energy of binding.
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D009391:Nephelometry and Turbidimetry; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014461:Ultracentrifugation
D000199:Actins; D015879:Myosin Subfragments; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D009218:Myosins