Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1.
Limited proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, and thermolysin yield fragments that correspond within 1-2K daltons to the 25K, 50K, and 20K fragments produced by trypsin. While papain and thermolysin cut preferentially at the 26K/70K junction, elastase and subtilisin cleave both the 26K/70K and the 75K/22K junctions in S-1. Using the above proteases as conformational probes, we have previously demonstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions [Applegate, D., & Reisler, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 7109-7112]. We report here that the binding of nucleotides at the active site is also sensed at both junctions. Both 2 mM MgADP and 5 mM MgATP slow the rate of elastase and subtilisin cleavage of the 95K heavy chain. With elastase, the 3-fold decrease in the rate of cleavage induced by nucleotides is evidenced at both the 26K/50K and the 50K/22K junctions. The analysis of subtilisin digestions is complicated by Mg nucleotide induced cleavage at a new site to produce a 91K fragment. Using N-methyl-6-anilinonaphthalene-2-sulfonyl chloride (MnsCl) to fluorescently label the 26K peptide, we demonstrate that the additional cleavage site is approximately 4K daltons from the N-terminal portion of the 95K heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011817:Rabbits; D013381:Subtilisins / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D015879:Myosin Subfragments; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010450:Endopeptidases; D013381:Subtilisins; D010196:Pancreatic Elastase; D014357:Trypsin; D010206:Papain; D013820:Thermolysin; D009218:Myosins; D008274:Magnesium