Bygrave F L, Daday A A, Doy F A
Biochem J. 1975 Mar;146(3):601-8. doi: 10.1042/bj1460601.
The EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-Ruthenium Red-quench technique (Reed & Bygrave, 1974a) was used to measure initial rates of Ca-2+ transport in mitochondria from flight muscle of the blowfly Lucilia cuprina. Evidence is provided for the existence in these mitochondria of a Ca-2+-transport system that has many features in common with that known to exist in rat liver mitochondria. These include requirement for energy, saturation at high concentrations of Ca-2+, a sigmoidal relation between initial rates of Ca-2+ transport and Ca-2+ concentration, a high affinity for free Ca-2+ (Km approx. 5 muM) and high affinity for the Ca-2+-transport inhibitoy, Ruthenium Red (approx. 0.03 nmol of carrier-specific binding-sites/mg of protein; Ki approx. 1.6 x 10- minus 8 M). Controlled respiration can be stimulated by Ca-2+ after a short lag-period provided the incubation medium contains KCl and not sucrose. The ability of Lucilia mitochondria to transport Ca-2+ critically depends on the stage of mitochondrial development; Ca-2+ transport is minimal in mitochondria from pharate adults, is maximal between 0 and 2h post-emergence and thereafter rapidly declines to reach less than 20% of the maximum value by about 2-3 days post-emergence. Respiration in mitochondria from newly emerged flies does not respond to added Ca-2+; that from 3-5-day-old flies is stimulated approx. 50%. Whereas very low concentrations of Ca-2+ inhibit ADP-stimulated respiration and oxidative phosphorylation in mitochondria from newly emerged flies (Ki approx. 60 ng-ions of Ca-2+/mg of protein); much higher concentrations (approx. 200 ng-ion/mg of protein) are needed to inhibit these processes in those from older flies. The potential of this system for studying the function and development of metabolite transport systems in mitochondria is discussed.
采用乙二醇双（乙胺）四乙酸（EGTA）-钌红淬灭技术（里德和拜格雷夫，1974a）来测定绿蝇丽蝇飞行肌线粒体中Ca²⁺转运的初始速率。有证据表明，这些线粒体中存在一种Ca²⁺转运系统，它具有许多与大鼠肝线粒体中已知的Ca²⁺转运系统相同的特征。这些特征包括对能量的需求、高浓度Ca²⁺时的饱和现象、Ca²⁺转运初始速率与Ca²⁺浓度之间的S形关系、对游离Ca²⁺的高亲和力（Km约为5 μM）以及对Ca²⁺转运抑制剂钌红的高亲和力（约0.03 nmol载体特异性结合位点/mg蛋白质；Ki约为1.6×10⁻⁸ M）。如果孵育培养基中含有KCl而非蔗糖，在短暂的延迟期后，Ca²⁺可刺激受控呼吸。丽蝇线粒体转运Ca²⁺的能力关键取决于线粒体的发育阶段；在预蛹期成虫的线粒体中Ca²⁺转运最少，在羽化后0至2小时之间最大，此后迅速下降，到羽化后约2至3天降至最大值的不到20%。新羽化苍蝇的线粒体呼吸对添加的Ca²⁺无反应；3至5日龄苍蝇的线粒体呼吸约被刺激50%。极低浓度的Ca²⁺会抑制新羽化苍蝇线粒体中ADP刺激的呼吸和氧化磷酸化（Ki约为60 ng Ca²⁺离子/mg蛋白质）；而抑制老龄苍蝇线粒体中的这些过程则需要更高的浓度（约200 ng离子/mg蛋白质）。讨论了该系统在研究线粒体中代谢物转运系统的功能和发育方面的潜力。
