The Conventional and Saturation Transfer Electron Paramagnetic Resonance of Spin-Labeled Myosin Subfragment-1 in the Presence of F-Actin and Nucleotides1

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Abstract
SH-1 thiol of S-1 was modified with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodo-acetoamide spin label (IASL). The extent of dissociation, a, of spin-labeled myosin subfragment-1 (IASL-S-1) from acto-IASL-S-1 by a nucleotide was measured by an ultracentrifugal separation method, a light-scattering method, and a saturation transfer EPR method. The a values obtained by these three methods were the same within the limits of the experimental errors. The dependence of a on the concentrations of AMPPNP, [S], and F-actin, [A], could be described by the equation: a−1=1+(1+KB/[S])[A]/KA. The KB and KA values were 0.65–1.2 mM and 1.7–2.7 mg/ml, respectively, in u.5 M KCl and 4 mM MgCl2 at pH 7.0 and 20°C. The height of the weakly immobilized peak of the conventional EPR spectrum of IASL-S-1, W, increased linearly with increase in the ATP or AMPPNP concentration, and became saturated at 1 mol nucleotide/mol IASL-S-1. No change in W was observed upon the binding of IASL-S-1 with F-actin. The dependence of the extent of change in W, Δ W, on [A] and [S] was given by ΔW−1=1+Ka/[S], where Ka = Ka / (1+KA/[A]). This finding indicates that the ΔW value is proportional to the amount of a nucleotide bound to IASL-S-1 and independent of the binding of F-actin to IASL-S-1.