An axoplasmic myosin with a calmodulin-like light chain.
Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximately 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 approximately 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.
Bearer
E L
EL
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912, USA.
DeGiorgis
J A
JA
Jaffe
H
H
Medeiros
N A
NA
Reese
T S
TS
D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D049832:Decapodiformes; D006737:Horseshoe Crabs; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins