PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF MYOMESIN, A MYOSIN-BINDING AND TITIN-BINDING PROTEIN, FROM BOVINE SKELETAL-MUSCLE

We report a method for isolating homogeneous myomesin from mammalian s keletal muscle. The identity of the purified bovine protein was confir med by its reactivity with myomesin-specific monoclonal antibodies and with polyclonal antibodies raised against peptides derived from the a mino-terminal and carboxy-tenxinal ends of the sequence predicted by t he human myomesin cDNA. All partial sequences obtained from bovine myo mesin can be aligned along the human sequence predicted by its cloned cDNA. Electron microscopy of myomesin revealed short flexible rods wit h a molecular length of about 50 nm. Circular dichroism spectra showed a high degree of beta structure as expected for a member of the immun oglobulin superfamily of proteins. Alignment of the sequences of the c lass I and II domains of myomesin with the sequences of domains of kno wn three-dimensional structure provides a more detailed model of myome sin. In agreement with this view, the cleavage sites observed by limit ed proteolysis locate primarily between individual domains. In a solid -phase overlay assay myomesin specifically bound to the myosin rod and to light meromyosin (LMM), but not to the carboxy-terminal 30-kDa fra gment of LMM. The myosin-binding site seemed to be confined to the ami no-terminal 240 residues of the molecule. The cross-reactivity of myom esin with the phosphorylation-dependent monoclonal neurofilament antib ody NE14 [Shaw, G. E., Debus, E. & Weber, K. (1984) Eur. J. Cell Biol. 34, 130-136] was analyzed. NE14 reactivity of myomesin was abolished by alkaline phosphatase. Reactivity of the antibody on stable proteoly tic fragments of myomesin showed that the phosphorylation site must re side within the carboxy-terminal 60 residues.