A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization

A new protein component of skeletal myofibrils has been isolated and characterized. It is prepared from impure myosin preparations and corresponds to band C, the principal contaminant observed in sodium dodecyl sulphate polyacrylamide gel electrophoresis patterns of such preparations (Starr and Offer, 1971).The C-protein, as we term it, is deduced to be a component of the skeletal myofibril because (i) glycerinated or fresh myoflbrils contain a component with a mobility identical to C-protein on sodium dodecyl sulphate gels, (ii) this component is extracted from myofibrils by the same solvent which extracts C-protein and (iii) C-protein may be prepared from preparations of isolated myofibrils. It is presumed to be a component of the thick filaments because it binds strongly to myosin at low ionic strength; immunological evidence which confirms this view is presented elsewhere.The quantity of C-protein in the myofibril has been estimated to be 2.0% by densitometry of sodium dodecyl sulphate gels of glycerinated myofibrils using actin as an internal reference. About forty molecules of C-protein are present in a thick filament.The properties of C-protein distinguish it from the other well-characterized myoflbrillar proteins. The C-protein molecule contains a single polypeptide chain of molecular weight 140,000. The intrinsic viscosity of 13.6 ml/g suggests that the molecule is neither completely globular nor as elongated as molecules like paramyosin or tropomyosin. The α-helical content is very low and the proline content higher than the other myofibrillar proteins. The molecule associates at low ionic strength.C-protein has no ATPase activity, nor does it affect the ATPase of pure myosin. But it reduces the activity of the actin-activated myosin ATPase by about half, this inhibition being independent of the level of Ca²⁺. C-protein does not bind Ca²⁺ in the presence of Mg²⁺. Its possible location and function are discussed.