Anthraniloyl ATP, a fluorescent analog of ATP, as a substrate for dynein ATPase and flagellar motility.
We synthesized an anthraniloyl ATP (ant-ATP), which has a fluorescent anthraniloyl moiety at the OH group of ribose, to elucidate the mechanism of flagellar bend formation and its propagation in relation to the mechanochemical cycle of dynein ATPase. This fluorescent analog of ATP was efficiently hydrolyzed by 21 S dynein from sea urchin sperm flagella with Km = 7.6 microM, whereas the Km was 12 microM when ATP was used as the substrate. Similar Vmax values were obtained with both ATP and ant-ATP. Inhibition of the hydrolysis of ant-ATP by vanadate was a little smaller than that with ATP. Photosensitized cleavage of 21 S dynein heavy chains in the presence of ant-ATP and vanadate was also a little less efficient than that in the presence of ATP and vanadate. Ant-ATP also induced the disintegration of the trypsin-treated axoneme and the motility of demembranated sperm in a manner similar to ATP. When ATP was used as a substrate for the demembranated sperm, the apparent Michaelis constant for beat frequency (Km f) was 0.22 mM and the maximum frequency (fmax) was 36 Hz, whereas Km f) was 0.14 mM and fmax was 20 Hz for ant-ATP. Thus ant-ATP could be an efficient fluorescent analog of ATP for studying dynein ATPase and the mechanisms of flagellar motility.
Inaba
K
K
Department of Biology, College of Arts and Sciences, University of Tokyo, Japan.
Okuno
M
M
Mohri
H
H
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D004398:Dyneins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D010777:Photochemistry; D012617:Sea Urchins; D013081:Sperm Motility; D013082:Sperm Tail / Q000502:physiology*; D013094:Spermatozoa / Q000502:physiology*; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000138:chemical synthesis / Q000378:metabolism*
D062367:ortho-Aminobenzoates; D014638:Vanadates; C045472:3'-O-anthraniloyl ATP; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins