Photocleavage of myosin subfragment 1 by vanadate.
The heavy chain of myosin's subfragment 1 (S1) was cleaved at two distinct sites (termed V1 and V2) after irradiation with UV light in the presence of millimolar concentrations of vanadate and in the absence of nucleotides or divalent metals. The V1 site cleavage appeared to be identical with the previously described active site cleavage at serine-180, which is effected by irradiation of a photomodified form of the S1-MgADP-Vi complex [Cremo, C. R., Grammer, J. C., & Yount, R. G. (1989) J. Biol. Chem. 264, 6608-6011]. The V2 site was cleaved specifically, without cleavage at the V1 site, first by formation of the light-stable S1-Co2+ADP-Vi complex at the active site [Grammer, J. C., Cremo, C. R., & Yount, R. G. (1988) Biochemistry 27, 8408-8415] and then by irradiation in the presence of millimolar vanadate. By gel electrophoresis, the V2 site was localized to a region about 20 kDa from the COOH terminus of the S1 heavy chain. From the results of tryptic digestion experiments, the COOH-terminal V2 cleavage peptide appeared to contain lysine-636 in the linker region between the 50- and 20-kDa tryptic peptides of the heavy chain. This site appeared to be the same site cleaved by irradiation of S1 (not complexed with Co2+ADP-Vi) in the presence of millimolar vanadate as previously described [Mocz, G. (1989) Eur. J. Biochem. 179, 373-378]. Cleavage at the V2 site was inhibited by Co2+ but was not significantly affected by the presence of nucleotides or Mg2+ ions. Tris buffer significantly inhibited V2 cleavage. From the results of UV-visible absorption, 51V NMR, and frozen-solution EPR spectral experiments, it was concluded that irradiation with UV light reduced vanadate +5 to the +4 oxidation state, which was then protected from rapid reoxidation by O2 by complexation with the Tris buffer. The relatively stable reduced form or forms of vanadium were not competent to cleave S1 at either the V1 or the V2 site. 51V NMR titration experiments indicated that a tetrameric species of vanadium preferentially bound to S1 and to the S1-MgADP-Vi complex, whereas no binding of either the monomeric or dimeric species could be detected. These results suggest that the vanadate tetramer was responsible for the photocleavage of S1 which occurred at both the V1 and V2 sites in the absence of nucleotides or divalent metals.
Cremo
C R
CR
Chemistry Department, Colorado College, Colorado Springs 80903.
Long
G T
GT
Grammer
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006868:Hydrolysis; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry; D015879:Myosin Subfragments / Q000378:metabolism*; D011817:Rabbits; D014466:Ultraviolet Rays; D014638:Vanadates / Q000378:metabolism*; D014639:Vanadium
D007554:Isotopes; D015879:Myosin Subfragments; D014639:Vanadium; D014638:Vanadates