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

Abstract

Myosin subfragment 1 (Sl) 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-= S C N -. The order of the inhibitiory effect is identical to the protein-structure-damaging effect of monovalent anions in the von Hippel series [von Hipel, P. (1 964) Scimce 145,577 -5811. '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 phosphatebinding 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, (Vi, 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.The interaction of myosin with actin and ATP generates the driving force of muscle contraction. 'The distinct ATPbinding and actin-binding sites of myosin reside on the globular head segment of the molecule. Due to the significant functional role of myosin-ATP and myosin-actin interactions, it is important to characterize the respective binding sites on myosin subfragment 1 (Sl). In spite of considerable efforts [l], the ATP-binding and actin-binding sites remain only partially characterized. According to studies which include limited tryptic digestion [2, 31, covalent cross-linking [4, 51 and the use of a specific antipeptide [6], at least part of the actinbinding site resides on the stretch at residues 632-643 of S1, at thejunction of the 50-kDa and 20-kDa tryptic S1 fragments Attempts to asign the ATP-binding site have been made using photoaffinity analogues of ATP [8 -111 and by analysis based on homology with other ATP-binding proteins [12. 131. On the basis of homology, a characteristic GXXXXGK sequence, corresponding to the stretch at residue...

Extensively Methylated Myosin Subfragment-1: Examination of Local Structure, Interactions with Nucleotides and Actin, and Ligand-Induced Conformational Changes

“…The impaired communication between the actin and nucleotide sites on M-Sl is also manifested in the lack of SI protection by actin from the Vi-dependent photocleavage at Ser-243, 31 kDa from the N-terminus, which is believed to be a part of the nucleotide-binding site of myosin (Grammer & Yount, 1991;Muhlrad et al, 1991Muhlrad et al, , 1992). The list of other methylation-related changes in the actin-triggered effects on SI includes (i) substantial if not complete loss of SI protection from heat treatment; (ii) loss of actin effects on thermolysin cleavage at the 25/50 kDa junction of SI; and (iii) reduced protection of SI from photocleavage by V; at 74 kDa from the N-terminus of actin, i.e., about 1 kDa upstream from the lysine-rich 50/20 kDa junction.…”

Distinct Topologies of Mono- and Decavanadate Binding and Photo-oxidative Cleavage in the Sarcoplasmic Reticulum ATPase

“…25). With regard to the mechanism of cleavage, it was reported that irradiation of the Mg ADPorthovanadate complex with myosin ATPase produces oxidation of a serine side chain to yield an aldehyde.…”

Vanadium and proteins: Uptake, transport, structure, activity and function

“…In addition to the biological applications of vanadate(V) to probe ATPases, another area of biomimetic chemistry of great interest is the vanadate(V)-induced photolytic cleavage of Vprotein complexes. The initial studies were carried out with dynein and myosin, which are ATPases providing energy for intracellular transport which cleave at one specific amino acid when exposed to UV light [341] [342] [343]. Since this early discovery, the photolytic cleavage of dynein, myosin, and adenosine kinase has been extensively studied, providing information on the phosphate-vanadate(V) binding site in the presence of free or bound nucleotides.…”