Effect of nucleotides, divalent cations and temperature on the tryptic susceptibility of myosin subfragment 1

The kinetics of tryptic breakdown of the heavy chain of chymotryptic myosin subfragment 1 (S1) according to the following scheme (where the numbers respresent approximate masses in kDa) are altered at 21°C by divalent cations (Me 2+ ) and by ATP, ADP, adenosine 5′‐[β, γ‐imino]triphosphate or PP i , with or without Me 2+ . ATP or its analogs slow step 2 and accelerate steps 3 and 4, while Me 2+ accelerates step 2. ATP and its analogs decrease the amount of a transient 27‐kDa peptide [Hozumi, T. & Muhlrad, A. (1981) Biochemistry 20 , 2945–2950]. We have found direct evidence for the suggestion in this reference that the 27‐kDa peptide is not an obligatory precursor of the 25‐kDa fragment and that ATP or ADP suppresses the formation of the larger N‐terminal fragment rather than accelerates its breakdown. Cross‐linking of sulfhydryl groups located in the 20‐kDa fragment leads to trapping of MgADP in the N‐terminal 25‐kDa peptide [Wells, J. A. & Yount, R. G. (1980) Biochemistry 19 , 1711–1717]; this process affects the tryptic fragmentation of S1 similarly to, but less effectively than, nucleotides. Acts‐S1 formation prevents the effect of ATP on fragmentation. At 37°C S1 loses ATPase activity; tryptic digestion proceeds more rapidly and the 50‐kDa and 25‐kDa fragments are degraded to small peptides. Nucleotides protect against the effects of higher temperature by producing conformational changes not only in the 27‐kDa N‐terminal portion (containing the putative nucleotide binding site) of the heavy chain of S1 but also in the 50‐kDa peptide.