Effects of Adenylyl Imidodiphosphate, a Nonhydrolyzable Adenosine Triphosphate Analog, on Reactivated and Rigor Wave Sea Urchin Sperm

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 μM ATP, and reactivated upon addition of 10-12 μM 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''≅ 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.