Organic anions stabilize the reactivated motility of sperm flagella and the latency of dynein 1 ATPase activity.

Abstract

Substitution of any of a variety of organic anions, including acetate, propionate, lactate, gluconate, and succinate, for chloride in the reactivation medium improves the motility of demembranated sperm of Tripneustes gratilla. At the optimum concentration of 0.20 N, all of these anions improve the duration of motility, with lactate and gluconate being the best. The Michaelis constant for beat frequency (Kmf) is lower (0.11-0.14 mM at 22 degrees C) in most of the organic anions than it is in Cl- (0.20 mM), and the minimum ATP concentration required to support oscillatory beating is reduced from 10 microM in chloride to 2 microM in acetate, which together indicate a greater affinity of the axonemal ATPase for MgATP2- in the organic anions media. The maximal beat frequency, fmax, is as high as 42 Hz in 0.2 N succinate compared to 31 Hz in Cl-, whereas the mean bend angle averages 2.8 rad in acetate compared to 2.4 rad in Cl-; these values give a calculated average velocity of tubule sliding of approximately 15 micron/s in acetate and succinate, which is approximately 30% greater than the value of 11 micron/s observed in chloride. The reactivated sperm are sixfold more sensitive to vanadate inhibition in 0.2 M acetate than they are in 0.15 M Cl-. The specific ATPase activity of soluble dynein 1, which increases more than 15-fold between 0 and 1.0 N Cl-, undergoes only a twofold activation over the same range of organic anion concentration, and, like the reactivated motility, is up to 50-fold more sensitive to vanadate. This greater apparent mechanochemical efficiency and the increased sensitivity to vanadate inhibition in the organic anions suggest that they, unlike chloride, do not promote the spontaneous dissociation of ADP and PO4(3-) from the dynein-ADP-PO4 kinetic intermediate in the dynein crossbridge cycle. The use of organic anion media may lead to significant improvements in reactivation of other motile and transport systems.