Studies on subfragment-I, a biologically active fragment of myosin.

The relationship between the structure and function of the myosin molecule is of considerable importance in the understanding of the molecular mechanism of muscle contraction. The nature of the adenosine triphosphatase (ATPase) site and the subunit structure are of special interest in this respect. The hydrolysis of ATP by myosin appears to link the conversion of chemical energy to the mechanical event of muscle contraction.' Sulfhydryl groups have long been implicated in this process and two unique sulfhydryl groups have been localized by specific chemical labeling methods.2-4 Reaction of one of these (Si) with thiol reagents results in an approximately sixfold increase in Ca++ATPase,5 whereas reaction of the second (S2) results in a loss of Ca++-ATPase.4 It has been suggested6 that both of these groups are located directly at the hydrolytic site of the enzyme. Myosin is composed of subunits that may be dissociated by guanidine or at alkaline pH.7 The myosin molecule would appear to comprise an axial core of two fibrous (f) or heavy subunits (mol wt 210,000) that terminate in a globular head containing three globular (g) or light subunits (mol wt 20,000).8-10 Although the Si and S2 sulfhydryl groups are present in the f-chains, the main core exhibits no residual ATPase activity following dissociation of the g-subunits.'0 Additional information concerning the substructure of myosin has been obtained by means of enzymatic digestion. Myosin may be degraded with trypsin" or insoluble papain'2 into a fragment of approximately 120,000 mol wt,'2-'7 termed subfragment-I,"1 which contains the full biological activity of the parent molecule. The number of subfragment-I particles produced from myosin has been controversial, with estimates ranging from one to three.'3-'5 17 Recent electron microscopy studies'8 suggest that the myosin molecule has a bipartite head, each lobe of which produces one subfragment-I particle. This report describes an investigation of the papainand trypsin-prepared subfragment-I molecules, with particular attention to (1) the presence of the Si and S2 sulfhydryls and (2) the subunit composition of these molecules. Materials and Methods.-Rabbit skeletal myosin was prepared according to a modification8 19 of the method of Szent-Gy6rgyi.20 The tryptic digestion procedure of Lowey and Cohen2' was used for the preparation of heavy meromyosin. Actin was extracted and purified according to the method of Carsten and Mommaerts.22 The insoluble papain complex (with cellulose) used in the preparation of subfragment-I was made according to the procedure of Lowey.23 Myosin was digested with the insoluble papain as described by Kominz, Mitchell, Nihei, and Kay.'2 The digestion of heavy meromyosin with trypsin was performed under the conditions described by Mueller and Perry." Subfragment-I was isolated as the F-actin-combining fraction" and was further purified by chromatography on Sephadex G-200 (Pharmacia).'4 The protein was concentrated by ultrafiltration under nitrogen pressure. Dissociation studies on subfragment-I were performed after overnight dialysis against 0.4 M KCl-0.1 M Na2C0a at pHI11.0.