|Budget Amount *help
¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1993 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1992 : ¥1,200,000 (Direct Cost : ¥1,200,000)
We attempt here to prepare and analyze several system composed of microtubule and dynein showing similar function to flagellar axonemes.
1) At first, we employed an in vitro assay system for sliding movement of doublet microtubules(DMts) of flagella along dynein-coated glass surface. Addition of ATP induced a circular sliding movement of the DMts with velocity of 2-4 um/sec. The movement depended upon concentrations ATP and Ca ions, which gave similar effects on flagellar axonemes in vivo.
2) Two kinds of complexes(Mts-dynein complexes) were prepared from ciliary 22S dynein and singlet microtubules reassembled from porcine brain tubulin. Both complexes were fully decorated with 22S dynein molecules. Addition of ATP induced sliding out of a microtubule from the complex, remaining a visible track. Sliding velocity of the microtubules on the dynein-track was about 12 um/sec, which was higher than that(8 um/sec) repported on the slide random-coated with dyneins(Vale & Toyoshima, 1988). Higher velocity may be due to the dynein-track, in which dynein molecules aligned with the same polarity in rows.
3) ATP-sensitive and -insensitive bindings have been reported in the microtubule-dynein complex. By addition of ATP to the complex, the amounts of dissociated and non-dissociated 22S dyneins were quantitatively analyzed, using gel-electrophoresis, measurement of liberated inorganic phosphate, and electron microscope. The results showed that about 90% of dynein in the complex was bound to microtubules in ATP-sensitive manner.