Subunit Structure of Myosin: III. A Proposed Model for Rabbit Skeletal Myosin

Abstract

Abstract High speed sedimentation equilibrium experiments indicate a molecular weight of 468,000 (±10,000) for rabbit skeletal myosin in 0.4 m KCl-0.05 m phosphate, pH 6.5. Weight average molecular weight values of 520,000 or above, which are presumably due to aggregation, are obtained in ultracentrifugal experiments on the same preparations of myosin. At pH 11, in 2 m guanidine, and on heat treatment, myosin is dissociated into a core of molecular weight 420,000 weight and light subunits of average molecular weight 20,200. The core may be dissociated by 5 m guanidine into two heavy subunits, of molecular weight 212,000 (±5,000), having no evident COOH-terminal group on digestion with carboxypeptidase A. The light alkali component comprises 11.7% (± 1%) of the protein, or 2.7 (±0.3) light subunits per myosin molecule, for unchromatographed myosin and myosin chromatographed once on diethylaminoethyl cellulose or cellulose phosphate; the molar ratio is reduced to 2.0 (±0.3) in a small fraction of the original myosin that is recovered after three chromatographic cycles on cellulose phosphate. Myosin may be reconstituted after subunit dissociation at alkaline pH or in guanidine solution; however, the reassociation of light subunits is diminished after prolonged denaturation. The removal of light subunits is accompanied by aggregation of the heavy chain core to low n-mers. In general, those denaturing conditions which lead to light subunit dissociation also result in the irreversible inactivation of adenosine triphosphatase and, on heat denaturation, values of ΔH‡ and ΔS‡ are comparable for both processes. The ATPase activity of myosin may be partially protected on alkaline treatment in 2 m KCl-0.01 m ATP; enzymic activity is lost in identically treated but fractionated heavy chain core and in augmented in the presence of excess light subunits.