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

Abstract

The kinetics of tryptic breakdown of the H chain of chymotryptic [rabbit muscle] myosin subfragment 1 (S1) according to the following scheme .**GRAPHIC**. (where the numbers represent approximate masses in kDa [kilodaltons]) are altered at 21.degree. C by divalent cations (Me2+) and by ATP, ADP, adenosine 5''-[.beta.,.gamma.-imino]triphosphate or PPi, with or without Me2+. ATP or its analogs slow step 2 and accelerate steps 3 and 4, while Me2+ accelerates step 2. ATP and its analogs decrease the amount of a transient 27-kDa peptide. Direct evidence was found for the suggestion 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 SH groups located in the 20-kDa fragment leads to trapping of MgADP in the N-terminal 25-kDa peptide; this process affects the tryptic fragmentation fo S1 similarly to, but less effectively than, nucleotides. Acts-S1 formation prevents the effect of ATP on fragmentation. At 37.degree. 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 H chain of S1 but also in the 50-kDa peptide.