Isolation and characterization of the 165 000 dalton protein component of the M-line of rabbit skeletal muscle and its interaction with creatine kinase.

The M-line protein component of molecular weight 165 000 was isolated and purified from rabbit skeletal muscle using ion exchange chromatography. Gel electrophoresis, in the presence and absence of sodium dodecyl sulfate, revealed the protein to be homogeneous. Sodium dodecyl sulfate gel electrophoresis and low speed sedimentation equilibrium studies in 0.5 M KCl, 50 mM potassium phosphate gave a molecular weight of 165 000 suggesting the protein to be made up of a single polypeptide chain. Circular dichroism spectra revealed the presence of two negative dichroic bands located at 216 and 208 nm, indicative of the presence of some beta-structure. Ellipticity values at these two wavelengths were --6500 +/- 400 and --7500 +/- 400 deg . cm2 . dmol-1, respectively. Addition of 165 000 component lowered the enzymatic activity of creatine kinase M-line protein and the nature of the inhibition was found to be a competitive one. When the protein was mixed with creatine kinase in a 1 : 1 mole ratio in a medium consisting of 0.2 M KCl, 25 mM Tris, 1 mM dithiothreitol (pH 8.0), low speed sedimentation equilibrium studies gave a molecular weight of 260 000 +/- 10 000 for the complex, indicative of an interaction of the two components of the M-line.