Two states of the conformation of 21S outer arm dynein coupled with ATP hydrolysis.
Tryptic digestion of 21S outer arm dynein from sea urchin sperm flagella in the presence of ATP (or ADP) and vanadate produced quite different polypeptides from those obtained in the absence of ATP (ADP) and/or vanadate (Inaba and Mohri (1989) J. Biol. Chem. 264, 8384-8388). The 21S dynein heavy chains were consistently digested into 165- and 135-kDa polypeptides in the absence of both ATP (ADP) and vanadate. In the presence of 2 mM ADP and 100 microM vanadate, 300-kDa polypeptide, which appeared to be a precursor of 165- and 135-kDa polypeptides, became less accessible to trypsin, and 165- and 135-kDa polypeptides were digested into 150-/148-kDa and 96-kDa polypeptides, respectively. Quantitative analysis of the degradation of 165- and 135-kDa polypeptides showed that the conformations of these polypeptides change remarkably in the presence of ATP (ADP) and vanadate, and slightly in the presence of ATP gamma S. Photoaffinity labeling with 8-azidoadenosine 5'-triphosphate and vanadate-mediated photocleavage of dynein heavy chains revealed that both adenine- and gamma-Pi-binding sites were located on 165- and 150-/148-kDa polypeptides, but not on 135-kDa polypeptide. These results suggest that the conformational change occurring in the 165-kDa region on binding ATP spreads to the 135-kDa region and causes the conformational change of the 135-kDa region.
Inaba
K
K
Department of Biology, College of Arts and Sciences, University of Tokyo.
Mohri
H
H
D000225:Adenine / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000032:analysis*; D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D004398:Dyneins / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007120:Immunochemistry; D010455:Peptides / Q000032:analysis; D010777:Photochemistry; D011487:Protein Conformation; D012617:Sea Urchins / Q000378:metabolism; D014357:Trypsin; D014638:Vanadates / Q000378:metabolism
D000345:Affinity Labels; D010455:Peptides; D014638:Vanadates; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D004398:Dyneins; D000225:Adenine