51V NMR study of vanadate binding to myosin and its subfragment 1.
The binding of various forms of vanadate to myosin and myosin subfragment 1 (S-1) was studied by 51V NMR at increasing vanadate concentrations between 0.06 and 1.0 mM. The distribution of the various forms of vanadate in the solution depended on the total concentration of vanadate. At low concentrations, the predominant vanadate form was monomeric, while at high concentration, it was tetrameric. The presence of myosin or S-1 in the solution produced a significant broadening of the signal of each form of vanadate, indicating that all of them bind to the protein. Addition of ATP, which does not affect the 51V NMR spectra in the absence of proteins, causes their significant alteration in the presence of myosin or S-1. The changes, which include the broadening of the signal of the monomeric and the narrowing of the signal of the oligomeric vanadate forms, indicate that more monomeric and less oligomeric vanadate binds to the proteins in the presence than in the absence of ATP. Irradiation by near-UV light in the presence of vanadate cleaves S-1 at three specific sites--at 23, 31, and 74 kDa from the N-terminus. The cleavages at 23 and 31 kDa are specifically inhibited by the addition of ATP. The vanadate-associated photocleavage of S-1 also depends on the total concentration of vanadate; it is observed only when the concentration of vanadate is at least 0.2 mM. This was also the lowest concentration at which oligomeric vanadate was detected in the 51V NMR spectra. From the parallel concentration dependence of the photocleavage and the appearance of the tetrameric vanadate, it is concluded that photocleavage occurs only when tetrameric vanadate binds to S-1.
Ringel
I
I
Department of Pharmacology, Hebrew University, Hadassah Medical School, Jerusalem, Israel.
Peyser
Y M
YM
Muhlrad
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009132:Muscles / Q000737:chemistry / Q000187:drug effects; D015879:Myosin Subfragments / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014638:Vanadates / Q000378:metabolism*
D007554:Isotopes; D015879:Myosin Subfragments; D014638:Vanadates; D000255:Adenosine Triphosphate; D009218:Myosins