Nucleotide-induced changes in the proteolytically sensitive regions of myosin subfragment 1

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Abstract
Limited proteolytic digestions of [rabbit muscle] myosin subfragment 1 (S-1) with elastase, subtilisin, papain and thermolysin yield fragments that correspond within 1-2 kilodaltons [K] 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, it was previously demonstrated that the binding of actin is sensed at both the 26K/50K and the 50K/22K junctions. The present report shows 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, the additional cleavage site was shown to be approximately 4K from the N-terminal portion of the 95K heavy chain. Despite the overall 2-fold decrease in the rate of cleavage of the 95K heavy chain, the rate of cleavage at the 50K/22K junction is increased 2-fold in the presence of nucleotides, and consequently the rate of cleavage at the 26K/50K junction must be decreased by a factor of 4. The binding of either 2 mM MgADP or 5 mM MgATP to S-1 caused a 2-fold increase in the rate of the thermolysin cleavage of S-1 at the 26K/70K junction but inhibited its cleavage by papain.