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- less than Cl- less than Br- less than 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-Vi (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.
Department of Oral Biology, Hadassah School of Dental Medicine, Jerusalem, Israel.
A
A
Muhlrad
Y M
YM
Peyser
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I
Ringel
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions; D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009218:Myosins / Q000378:metabolism* / Q000528:radiation effects; D010777:Photochemistry; D011817:Rabbits; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology*
D000838:Anions; D002413:Cations, Divalent; D014638:Vanadates; D000255:Adenosine Triphosphate; D009218:Myosins