Studies on the chymotryptic digestion of myosin. Effects of divalent cations on proteolytic susceptibility

Abstract

Experiments have been carried out to explore the specificity of proteolytic cleavage of rabbit skeletal muscle myosin by chymotrypsin. Whilst heavy meromyosin is obtained most readily from monomeric myosin in 0·6 m -NaCl, polymeric myosin in 0·12 m -NaCl also yields heavy meromyosin when digested in the presence of divalent cations. Digestion of polymeric myosin in the absence of divalent cations produces subfragment-1. Thus the two regions of myosin susceptible to proteolytic cleavage may be discriminated by the presence or absence of divalent cations when polymeric myosin is exposed to brief chymotrytic digestion. This metal dependence suggests that the flexibility of the two sites is modulated by divalent cations. Calcium ions are more effective than magnesium ions in protecting the subfragment-1 site. This protective effect appears to be due to the presence of the DTNB light chains, which are known to bind calcium ions. Chymotryptic digestion of isolated DTNB light chains or myosin shows that these light chains are readily susceptible to proteolysis, and the specific site of proteolytic attack varies when divalent cations are present. The two principal sites of cleavage within the DTNB light chain have been identified by N-terminal analysis, and these sites are located on either side of the putative calcium binding site, identified from the amino acid sequence ( Collins, 1976 ). Brief digestion of polymeric myosin in the absence of divalent cations shows that the DTNB light chains are cleaved more readily, than the subfragment-1 site in the heavy chains. Thus these light chains become degraded in myosin molecules with uncleaved heavy chains. Further digestion of this material in the presence of divalent cations shows no protection of the subfragment-1 site. DTNB light chains cleaved in myosin that has been digested with chymotrypsin in the presence of divalent cations still protect the subfragment-1 site during furthur exposure to chymotrypsin. These results suggest that protection of the subfragment-1 site requires the presence of DTNB light chains with intact calcium binding sites. The different proteolytic fragments obtained from myosin by chymotryptic cleavage have been identified by polyacrylamide gel electrophoresis and their apparent molecular weights are discussed in relation to myosin fragmentation products obtained with other proteinases.