Rapid method for isolation of myosin subfragment-1 from carp myofibrils.

A rapid method for isolation of myosin subfragment-1 (S-1) from carp myofibrils (Mf) was developed. For preparation of S-1, Mf were digested by α-chymotrypsin in a medium of 0.05M KCI (pH 7.0) containing lmM EDTA, cleaving at a head-tail junction of the myosin molecule. S-1 was dissociated from F-actin upon Mg-pyrophosphate addition. Crude S-1 was obtained in the supernatant after centrifugation, then purified by (NH4)2SO4 fractionation be-tween 40 and 50% saturation, and by Sephacryl S-300 gel filtration. Since the amount of S-1 produced from thermally treated Mf was proportional to the remaining Ca-ATPase activity of Mf, it was concluded that S-1 was produced only from native myosin contained in Mf. Therefore, partially denatured muscle such as frozen or stored muscle can be used as a starting material for S-1 preparation. This also suggests the possibility that the native myosin content in muscle can be estimated from the amount of S-1 produced by the chymotryptic digestion of Mf.