Vanadate-mediated photocleavage of rabbit skeletal myosin.
The heavy chain of myosin from rabbit skeletal muscle can be cleaved at three sites by irradiation with near-ultraviolet light in the presence of 0.1-1.0 mM vanadate. The sigmoidal dependence upon vanadate concentration, with half-maximal rate occurring at about 0.5 mM vanadate and a sigmoidicity of 2.7, is consistent with the chromophore responsible for cleavage being oligomeric vanadate. Cleavage occurs at two sites located within the head region of the molecule, 23 kDa and 75 kDa from the NH2-terminus; these sites are cleaved equally well in heavy meromyosin and subfragment 1. In the presence of 1 mM vanadate, the half-times for cleavage of the 23-kDa and 75-kDa sites are about 15 and 10 min, respectively. The rate of cleavage at both these sites is retarded 2-3-fold by the presence of greater than 10 microM MgATP. The third photocleavage site is located about 5-10 kDa from the COOH terminus of the intact heavy chain, and cleaves equally well in the isolated rod and in light meromyosin. Cleavage at this site occurs with a half-time of 138 min, and its rate is unaffected by the presence of MgATP. The vanadate-mediated cleavage of the heavy chains is accompanied by characteristic changes in the myosin ATPase properties, with the Ca2+, Mg2+ and actin-activated Mg2+ ATPases becoming elevated, whereas the K+/EDTA ATPase becomes inactivated. The sites of photocleavage in the myosin heavy chain might be associated with sites of phosphate binding.
Mocz
G
G
Pacific Biomedical Research Center, University of Hawaii, Honolulu.
D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D015879:Myosin Subfragments / Q000528:radiation effects; D009218:Myosins / Q000378:metabolism / Q000528:radiation effects*; D010446:Peptide Fragments / Q000528:radiation effects; D010777:Photochemistry; D010782:Photolysis; D011817:Rabbits; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology*
D015879:Myosin Subfragments; D010446:Peptide Fragments; D014638:Vanadates; D009218:Myosins