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. 1978 Jan;75(1):54–58. doi: 10.1073/pnas.75.1.54

Formation of a ternary complex: Actin, 5′-adenylyl imidodiphosphate, and the subfragments of myosin

Lois E Greene 1, Evan Eisenberg 1
PMCID: PMC411182  PMID: 343111

Abstract

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°. With 40 μM 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.

Keywords: myosin subfragment 1, heavy meromyosin, association constants, turbidity

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Selected References

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