An Axoplasmic Myosin with a Calmodulin-Like Light Chain

• Published in: Proceedings of the National Academy of Sciences - PNAS 1996-06, Vol.93 (12), p.6064-6068
• Main Authors: Bearer, Elaine L., DeGiorgis, Joseph A., Jaffe, Howard, Medeiros, Nelson A., Reese, Thomas S.
• Format: Article
• Language: English
• Subjects: Actins; Adenosine triphosphatases; Amino Acid Sequence; Amino acids; Animals; Antibodies; Axons; Axons - metabolism; Base Sequence; Blotting, Western; Ca(2+) Mg(2+)-ATPase - metabolism; Calmodulin - chemistry; Calmodulin - genetics; Calmodulin - metabolism; Cloning, Molecular; Cytosol; Decapodiformes; Gels; Horseshoe Crabs; Limulus polyphemus; Loligo pealei; Marine; Marine biology; Molecular Sequence Data; Molecular weight; Myosins - metabolism; Neurology; Organelles; Organelles - metabolism; Proteins; Sequence Homology, Amino Acid; Sequencing
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.93.12.6064

Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An ≈ 235-kDa protein, the only band recognized by a pan-myosin antibody in Western blots of isolated axoplasmic organelles, has been previously proposed to be a motor for these movements. Here, we purify this ≈ 235-kDa protein (p235) from axoplasm and demonstrate that it is a myosin, because it is recognized by a pan-myosin antibody and has an actin-activated Mg-ATPase activity per mg of protein 40-fold higher than that of axoplasm. By low-angle rotary shadowing, p235 differs from myosin II and it does not form bipolar filaments in low salt. The amino acid sequence of a 17-kDa protein that copurifies with p235 shows that it is a squid optic lobe calcium-binding protein, which is more similar by amino acid sequence to calmodulin (69% identity) than to the light chains of myosin II (33% identity). A polyclonal antibody to this light chain was raised by using a synthetic peptide representing the calcium binding domain least similar to calmodulin. We then cloned this light chain by reverse transcriptase-PCR and showed that this antibody recognizes the bacterially expressed protein but not brain calmodulin. In Western blots of sucrose gradient fractions, the 17-kDa protein is found in the organelle fraction, suggesting that it is a light chain of the p235 myosin that is also associated with organelles.