Preparation of a myosin-extracted "ghost" myofibril Sephadex conjugate column and its application to the separation of myosin subfragment-1 giving and not giving the initial burst of phosphate.
A "ghost" myofibril (myosin-extracted myofibril) Sephadex conjugate which specifically binds myosin, HMM and S-1 in the absence of Mg-ATP or Mg-PP can be prepared in a few days by conjugating "ghost" myofibrils to Sephadex beads. Binding ability is retained for over a month. It is used, therefore, for actin-affinity chromatography of myosin and its active fragments. It is under debate whether the two heads of the myosin molecule are functionally identical. Recently several reports have indicated that S-1 could be separated into two kinds of S-1, one giving the initial burst of phosphate and the other not, by assuming a difference in the affinity of the two kinds of S-1 to F-actin. Attempts are reported here to obtain these two components of S-1 separately by using the "ghost" myofibril Sephadex conjugate column. The method of S-1 separation reported by Shibata-Sekiya and Tonomura ((1976) J. Biochem, 80, 1371-1380), which used S-1 treated with CMB, was applied to the "ghost" myofibril Sephadex conjugate column. This resulted in the successful separation of S-1 modified with CMB giving no initial burst of phosphate and unmodified S-1 giving the initial burst of phosphate. A separation method based essentially on the principle employed by Taniguichi and Tawada ((1976) J. Biochem. 80, 853-860) gave an unsuccessful result.
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000166:cytology*; D009210:Myofibrils / Q000378:metabolism / Q000648:ultrastructure; D015879:Myosin Subfragments / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits
D000199:Actins; D015879:Myosin Subfragments; D010710:Phosphates; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium