| Research Abstract |
The motor protein responsible for cytoplasmic streaming in Characean cells (Chara myosin) can slide actin filaments at the velocity of 60mum/s, which is ten times faster than the sliding movement of skeletal muscle myosin. We succesfully purified this Chara motor protein, Chara myosin, heavily contaminated proteases, and found some distinct characterstics of this myosin, in addition to the fastest sliding movement, and reported the results (reference 1).
In order to get clue for elucidating the molecular basis of this distinct function of Chara myosin, we examined by using various techniques, particularly molecular biology techniques for cloning and sequencing of Chara myosin.
It was very difficult to purify mRNA from Characean cells, which contained endoplasm only 1% of the total cell volume. However, we improved the mRNA purification method and fortunately got mRNA with sufficient purity, although quantity was very small. We made cDNA library for cloning of the fastest Chara myosin. Th
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e probes for screening of the cDNA library were obtained from PCR using primers designed according to peptide microsequences of the Chara myosin and also consensus sequences of various myosins. The product of PCR contained 5 clones, each of which showed great similarity to any of known myosins. We are now continuing to analyze the clones obtained and the structure of Chara myosin.
So far, the sliding velocity is understood to correlate with the actomyosin ATPase activity. We found, however, that the velocity did not correlated with the actomyosin ATPase activity, but strongly correlated with the reciprocal of the ATPase activity of myosin itself, which corresponds to the life time of the intermediate state of the rate limitting step of the myosin ATPase cycle, myosin ADP Pi. The longer the life time of myosin ADP Pi, the faster the sliding velocity. Therefore, we can predict that the fastest Chara myosin must have much longer life time at the intermediate state than any other myosins. This finding can promisingly lead us to the clue to elucidate the molecular mechanism of the tremendously fast velocity of Chara myosin. Less
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