Preparation of Highly Purified Myosin from Mantle Muscle of Squid, Ommastrephes sloani pacificus

Abstract

1) Myosin was isolated from the squid mantle muscle in a high yield by EBASHI's method which has been used for preparation of chicken gizzard myosin. Paramyosin contaminating in the preparation, was successfully eliminated by the following procedure: rabbit F-actin was added to form acatomyosin complesx with squid myosin in the medium containing 0.5M KCI. Acatomyosin formed was sedimented by ultracentriafugation, leaving paramyosin in the supernatant. The pellet thus obtained was dissolved in 0.5M KCI (pH 7.0) and the pure myosin was collected by salting out with (NH₄)₂SO₄ in the presence of ATP-Mg.
2) The squid myosin thus prepared was shown to be homogeneous on ultracentrifugal sedimentation. The sedimentation constant (s_20.w) was estimated to be 6.0 S corresponding to the monomeric myosin.
3) The SDS-polyacarylamide gel electrophoretic patterns showed that the squid myosin contained protein componets corresponging to a heavy chain and two light chains with molecular weights of 15, 000 and 17, 000.
4) The ATPase activity of squid myosin was different from that of rabbit skeletal myosin with respect to specific avativity and KCI concentration dependence.
5) The squid myosin was similar to that of rabbit myosin in acatin avtivation of its Mg-ATPase activity.
6) The Ca-ATPase avtivity of squid myosin was approximatedy fifty times sensitive to heat-inavtivation, like that of rabbit myosin.