An axoplasmic myosin with a calmodulin-like light chain

被引:13
作者
Bearer, EL
DeGiorgis, JA
Jaffe, H
Medeiros, NA
Reese, TS
机构
[1] BIOL MARINE LAB,WOODS HOLE,MA 02543
[2] NINCDS,NIH,PEPTIDE SEQUENCING FACIL,NEUROCHEM LAB,BETHESDA,MD 20892
[3] NINCDS,NIH,NEUROBIOL LAB,BETHESDA,MD 20892
关键词
D O I
10.1073/pnas.93.12.6064
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Organelles in the axoplasm from the squid giant axon move along exogenous actin filaments toward their barbed ends. An approximate to 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 approximate to 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 bq 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.
引用
收藏
页码:6064 / 6068
页数:5
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