Dynamic conformational changes of 21 S dynein ATPase coupled with ATP hydrolysis revealed by proteolytic digestion.
Conformational changes of 21 S dynein ATPase from sea urchin sperm flagella were examined by tryptic digestion under physiological conditions. In the presence of 2 mM ATP or ADP plus 100 microM inorganic vanadate (Vi), dynein heavy chains were digested by trypsin into quite different polypeptides from those obtained in other cases (no addition, 2 mM ATP, 4 mM adenosine 5'-(beta,gamma-imido)triphosphate, 4 mM adenosine 5'-(beta,gamma-methylene)triphosphate, 2 mM ADP, 100 microM Vi). In the presence of 4 mM adenosine 5'-O-(3-thiotriphosphate), however, the digestion pattern was similar to that in the presence of ATP (ADP) and Vi, to a certain extent. In all conditions other than the presence of ATP (ADP) and Vi, 165- and 135-kDa polypeptides were the main products, whereas in the presence of ATP (ADP) and Vi, 200-, 150/148-, and 105/96-kDa peptides were produced and 320-kDa peptide became rather inaccessible to trypsin. The latter digestion pattern was not observed in the absence of divalent cations. These results suggest that, in the ATP hydrolysis cycle, dynein changes its conformation remarkably in the dynein-ADP-Pi state, which is presumably responsible for force generation.
Inaba
K
K
Department of Biology, College of Arts and Sciences, University of Tokyo, Japan.
Mohri
H
H
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent; D002918:Chymotrypsin / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D012617:Sea Urchins; D012697:Serine Endopeptidases / Q000378:metabolism; D013094:Spermatozoa / Q000201:enzymology; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D002413:Cations, Divalent; D010446:Peptide Fragments; C022571:adenosine 5'-O-(3-thiotriphosphate); D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D002918:Chymotrypsin; C028344:glutamyl endopeptidase; D014357:Trypsin; D013820:Thermolysin; D000251:Adenosine Triphosphatases; D004398:Dyneins