The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella.
The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.
Inoue
Yuichi
Y
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo, Japan.
Shingyoji
Chikako
C
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D004398:Dyneins / Q000378:metabolism*; D008297:Male; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D013082:Sperm Tail / Q000378:metabolism*; D047269:Strongylocentrotus / Q000378:metabolism*
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004398:Dyneins