Solubilization of dynein from Tetrahymena ssp. axonemes using phosphate analogues.
One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp. (T. pyriformis or T. thermophila) in the presence of ATP. This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein. Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins. These results suggest that the solubilized protein is an outer arm dynein and also that a dynein-ADP-phosphate complex decreases its affinity with the adjacent microtubules within axonemes. Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue. The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.
Nakamura
Ken-ichi
K
Department of Health Sciences, Hiroshima Prefectural Women's University, Ujina-higashi, 1-1-71, Minami-ku, Hiroshima 734-8558, Japan. nakamura@hirojo-u.ac.jp
Tanaka
Miwa
M
D000251:Adenosine Triphosphatases / Q000378:metabolism; D017607:Aluminum Compounds / Q000737:chemistry; D000818:Animals; D001608:Beryllium / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry; D002923:Cilia / Q000378:metabolism*; D004398:Dyneins / Q000737:chemistry / Q000302:isolation & purification*; D005459:Fluorides / Q000737:chemistry; D008870:Microtubules / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D013768:Tetrahymena / Q000378:metabolism*; D014466:Ultraviolet Rays; D014638:Vanadates / Q000737:chemistry
D017607:Aluminum Compounds; D021122:Protein Subunits; D014638:Vanadates; C035982:beryllium fluoride; D002918:Chymotrypsin; D000251:Adenosine Triphosphatases; D004398:Dyneins; D001608:Beryllium; D005459:Fluorides; C032311:aluminum fluoride