A nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), has been used to study the role of ATP binding in flagellar motility. Sea urchin sperm of Lytechinus pictus were demembranated, reactivated, and locked in "rigor waves" by a modification of the method of Gibbons and Gibbons (11). Rigor wave sperm relaxed within 2 min after addition of 4 micrometer ATP, and reactivated upon addition of 10-12 micrometer ATP. The beat frequency of the reactivated sperm varied with ATP concentration according to Michaelis-Menten kinetics ("Km" = 0.24 mM; "Vmax" = 44 Hz) and was competitively inhibited by AMP-PNP (Ki" approximately to 8.1 mM). Rigor wave sperm were completely relaxed (straightened) within 2 min by AMP-PNP at concentrations of 2-4 mM. The possibilities that relaxation in AMP-PNP was a result of ATP contamination, AMP-PNP hydrolysis, or lowering of the free Mg++ concentration were conclusively ruled out. The results suggest that dynein cross-bridge release is dependent upon ATP binding but not hydrolysis.
Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm.
S M Penningroth, G B Witman; Effects of adenylyl imidodiphosphate, a nonhydrolyzable adenosine triphosphate analog, on reactivated and rigor wave sea urchin sperm.. J Cell Biol 1 December 1978; 79 (3): 827–832. doi: https://doi.org/10.1083/jcb.79.3.827
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