ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein.
Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (A alpha, A beta, and A gamma) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained A beta and A gamma HCs, while the A alpha HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887-895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897-901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 micrograms/ml thermolysin for 45 min, we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5 alpha, 5 beta, 6 alpha, and 6 beta). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained A beta or A gamma HC. Thermolysin digests, TH 1, 2, 5 alpha and 6 beta were found to be derived from A beta HC, while TH 3, 4, 5 beta and 6 alpha originated in the A gamma HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the A beta and A gamma HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.
Niino
Y
Y
Department of Biology Ochanomizu University, Tokyo, Japan.
Miki-Noumura
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D002923:Cilia / Q000201:enzymology*; D004398:Dyneins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010449:Peptide Mapping; D013768:Tetrahymena / Q000201:enzymology*; D013820:Thermolysin
D002918:Chymotrypsin; D013820:Thermolysin; D000251:Adenosine Triphosphatases; D004398:Dyneins