Effects of ions on vanadate‐induced photocleavage of myosin subfragment 1

Myosin subfragment 1 (S1) is cleaved by near‐ultraviolet irradiation in the presence of vanadate at three sites located at 23, 31 and 74 kDa from the N‐terminus. Since vanadate is considered to be a good structural analogue of phosphate, it is assumed that the cleavage sites participate in forming the phosphate‐binding site(s) of S1. In this work, the effect of various ions on the vanadate‐induced photocleavage of S1 was studied. Monovalent anions were found to inhibit photocleavage in the 50–200 mM range. The inhibition is more expressed at a site 74 kDa from the N‐terminus than at the 23‐kDa and 31‐kDa sites. The inhibitory effect of the monovalent anions increases in the order acetate = F⁻ < CI⁻ < Br⁻ < I⁻= SCN⁻. The order of the inhibitory effect is identical to the protein‐structure‐damaging effect of monovalent anions in the von Hippel series [von Hipel, P. H. & Wong, K. Y.(1964) Science 145, 577–581]. Therefore, it is assumed that decreased photocleavage is due to local perturbations of structure, especially at the 74‐kDa site, in addition to increased ionic strength. Divalent anions, sulfate and thiosulfate, strongly inhibit photocleavage at 2 mM. The inhibition is very pronounced at the 23‐kDa and 31‐kDa sites, while the 74‐kDa site is hardly affected. Since photocleavage at the 23‐kDa and 31‐kDa sites is regulated jointly and independently from cleavage at the 74‐kDa site, it is assumed that S1 has two distinct phosphate‐binding sites: the regions of the 23‐kDa and 31‐kDa cleavage sites, which are proximal to each other in the spatial structure, participate in forming the first phosphate‐binding site, while the 74‐kDa site is part of the second binding site. Sulfate was also found to inhibit the trapping of vanadate and to facilitate its release from the S1‐MgADP‐V_i (V_i, inorganic vanadate) complex. Photocleavage of S1 takes place at all three sites, both in the presence or absence of divalent cations, indicating that these, including Mg²⁺, are not essential for cleavage.