Gibbons B H, Tang W J, Gibbons I R
J Cell Biol. 1985 Oct;101(4):1281-7. doi: 10.1083/jcb.101.4.1281.
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.
在激活介质中用包括乙酸盐、丙酸盐、乳酸盐、葡萄糖酸盐和琥珀酸盐在内的多种有机阴离子中的任何一种替代氯化物，可提高海胆（Tripneustes gratilla）去膜精子的活力。在0.20 N的最佳浓度下，所有这些阴离子都能延长活力持续时间，其中乳酸盐和葡萄糖酸盐效果最佳。大多数有机阴离子中，用于搏动频率的米氏常数（Kmf）（22℃时为0.11 - 0.14 mM）低于Cl-中的米氏常数（0.20 mM），且支持振荡搏动所需的最低ATP浓度从氯化物中的10 μM降至乙酸盐中的2 μM，这共同表明在有机阴离子介质中轴丝ATP酶对MgATP2-的亲和力更高。在0.2 N琥珀酸盐中，最大搏动频率fmax高达42 Hz，而在Cl-中为31 Hz；相比之下，乙酸盐中的平均弯曲角度平均为2.8弧度，Cl-中为2.4弧度；这些值计算得出乙酸盐和琥珀酸盐中小管滑动的平均速度约为15微米/秒，比氯化物中观察到的11微米/秒的值大约高30%。在0.2 M乙酸盐中，再激活的精子对钒酸盐抑制的敏感性比在0.15 M Cl-中高六倍。可溶性动力蛋白1的比ATP酶活性在0至1.0 N Cl-之间增加超过15倍，在相同的有机阴离子浓度范围内仅经历两倍的激活，并且与再激活的活力一样，对钒酸盐的敏感性高达50倍。这种更高的表观机械化学效率以及在有机阴离子中对钒酸盐抑制的敏感性增加表明，与氯化物不同，它们不会促进动力蛋白 - ADP - PO4动力学中间体在动力蛋白横桥循环中自发解离ADP和PO4(3-)。使用有机阴离子介质可能会显著改善其他运动和运输系统的再激活。
