Interactions of the actin and nucleotide binding sites on myosin subfragment 1.

S Highsmith

doi:10.1016/s0021-9258(20)81839-0

Abstract

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.

Highlights

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-l) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCI, pH 7.0 at 4”
Nucleotide concentrations were varied between 2 and 200 x lo-‘M. These concentrations of S-1 and actin are as close to infinite dilution as could be measured conveniently
Association Constants-The association constants reported here for nucleotide binding to S-l are in reasonable agreement with those obtained by other workers, critical comparisons are difficult because of the differences in conditions and protein preparations

Summary

Introduction

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-l) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCI, pH 7.0 at 4”. For ADP, K’, was 2.7 x 10’ Mm’ and the interaction free energy was -4.67 kJ M-l. A ternary complex was shown to exist for ADP, S-l, and actin in the presence of Mg*+ and evidence from AMP-P(NH)P and PP, measurements indicated that ATP likely forms a ternary complex. There is some controversy as to whether the nucleotide and actin binding sites are separate or overlapping. Based on the effects of thiol modification of myosin, BBr6ny and B&Any have claimed that the sites are separate (l), while Harrington et al [2] using related methods have indicated that the sites overlap

Methods

Results

Conclusion