Effect of nucleotides, actin and temperature on thermolysin digestion of myosin subfragment-1.
Myosin subfragment-1 from rabbit skeletal muscle was digested by thermolysin at 25 degrees, 12 degrees and 0 degree C. Thermolysin cleaves subfragment-1 heavy chain into two stable fragments, 28 kDa and 70 kDa, aligned in this order from the N-terminus [Applegate, D. & Reisler, E. (1983) Proc. Natl Acad. Sci. USA 80, 7109-7112]. The rate of digestion at 25 degrees C was significantly increased in the presence of MgATP and somewhat less in the presence of MgADP, or magnesium pyrophosphate. This activating effect of the nucleotides was decreased at 12 degrees C and completely eliminated at 0 degrees C. The results can be explained by assuming that there are two subfragment-1 conformers [Shriver, J. W. & Sykes, B. D. (1981) Biochemistry 20, 2004-2012], and that both the addition of ATP or its analogs, and lowering the temperature, shift the conformational equilibrium in the direction that is more susceptible to thermolysin. Actin inhibited thermolysin digestion of subfragment-1 at all three temperatures studied. Actin inhibition can be explained either by shifting the equilibrium of the conformers in the direction of the less susceptible form or by direct interference of actin with the binding of thermolysin to subfragment-1. Actin inhibition of thermolysin digestion also prevailed when subfragment-1 was in a ternary complex with nucleotide and actin, in both the strongly and weakly attached states. Similarly, actin inhibited the digestion of subfragment-1 modified by 4-phenylenedimaleimide [corrected], which also forms a weakly attached complex with actin. No difference could be found in the accessibility of the thermolysin-susceptible site of subfragment-1 at the 28-70 kDa junction in either rigor, strongly or weakly attached states, which indicates the similarity of the structure proximal to this specific site in the three attached states.
Cardiovascular Research Institute, University of California, San Francisco.
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Muhlrad
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Chaussepied
D000199:Actins / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000737:chemistry; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology*; D011817:Rabbits; D013696:Temperature; D013820:Thermolysin / Q000494:pharmacology*
D000199:Actins; D015879:Myosin Subfragments; D009711:Nucleotides; D013820:Thermolysin; D008274:Magnesium