Gassner D, Komnick H
Eur J Cell Biol. 1981 Aug;25(1):108-19.
An anion-stimulated, Mg2+-dependent, ouabain-insensitive ATPase is present in salt gland homogenates of domestic ducks (Anas platyrhynchos). The enzyme is unspecifically stimulated by various inorganic and organic anions including amino and sulfonic acids which are often used as buffer components (e. g. histidine, Bicin, PIPES, MES and HEPES). Therefore, the demonstration of ATPase stimulation by chloride strongly depends on the type and concentration of the buffer used and may also largely interfere with the stimulation caused by other anions present in the incubation medium. Of the inorganic anions tested chloride and bicarbonate appear to be the favorite physiological activators, but the possible role of carbonic acids in the stimulation of the anion-dependent ATPase should not be neglected. Km values are approximately 5.8 mM for Cl- and approximately 8.7 mM for HCO-3-activation. Maximal ATPase stimulation is obtained at 25 mM Cl- and approximately 30 mM HCO-3, respectively. The simultaneous presence of bicarbonate decreases chloride affinity and Vmax, and shifts the chloride optimum to lower concentrations. ATP is the most preferred substrate. Maximal activation by Cl- and HCO-3 occurs at ATP concentrations between 0.5 and 1 mM. ATP affinity increases in the presence of Cl- and HCO-3, respectively. Both chloride and bicarbonate require a Mg2+ to ATP ratio of approximately 0.5 and a pH value of 8.0 to 8.5 for optimal stimulation. Stimulation by Cl- and HCO-3 is inhibited by thiocyanate, cyanate and by the diuretic drugs furosemide, ethacrynic acid and mersalyl. Incubation media adapted for the simultaneous demonstration of both chloride and bicarbonate activation contained 10 to 20 mM histidine-Tris buffer at pH 8.0 to 8.5, 150 mM sucrose, 0.2 mM ouabain, 0.5 mM magnesium acetate, 1 mM ATP, (pH adjusted to 8.0-8.5 with Tris or NaOH), with and without 25 mM sodium chloride or 25 mM sodium bicarbonate.
在家鸭（绿头鸭）的盐腺匀浆中存在一种阴离子刺激、Mg2+依赖、哇巴因不敏感的ATP酶。该酶受到各种无机和有机阴离子的非特异性刺激，包括经常用作缓冲成分的氨基酸和磺酸（例如组氨酸、Bicin、PIPES、MES和HEPES）。因此，氯离子对ATP酶的刺激作用很大程度上取决于所用缓冲液的类型和浓度，也可能在很大程度上干扰孵育介质中其他阴离子引起的刺激。在所测试的无机阴离子中，氯离子和碳酸氢根似乎是最主要的生理激活剂，但碳酸在刺激阴离子依赖性ATP酶方面的可能作用不应被忽视。氯离子激活的Km值约为5.8 mM，碳酸氢根激活的Km值约为8.7 mM。分别在25 mM氯离子和约30 mM碳酸氢根时可获得最大ATP酶刺激。碳酸氢根的同时存在会降低氯离子的亲和力和Vmax，并将氯离子的最佳浓度范围移至较低浓度。ATP是最优选的底物。氯离子和碳酸氢根在ATP浓度为0.5至1 mM之间时发生最大激活。分别在氯离子和碳酸氢根存在的情况下，ATP亲和力增加。氯离子和碳酸氢根都需要Mg2+与ATP的比例约为0.5，pH值为8.0至8.5才能实现最佳刺激。氯离子和碳酸氢根的刺激作用受到硫氰酸盐、氰酸盐以及利尿药速尿、依他尼酸和汞撒利的抑制。适用于同时显示氯离子和碳酸氢根激活作用的孵育介质含有pH值为8.0至8.5的10至20 mM组氨酸 - Tris缓冲液、150 mM蔗糖、0.2 mM哇巴因、0.5 mM乙酸镁、1 mM ATP（用Tris或NaOH将pH值调至8.0 - 8.5），添加或不添加25 mM氯化钠或25 mM碳酸氢钠。
