Kolomiiets' O V, Danylovych Iu V, Danylovych H V, Kosterin S O
Ukr Biokhim Zh (1999). 2013 Jul-Aug;85(4):30-9. doi: 10.15407/ubj85.04.030.
The opportunity of Ca2+-sensitive fluorescent dye Fluo-4 AM and spectrofluorimetry method application for the study of energy-dependent Ca2+ accumulation in mitochondria from uterus smooth muscle is proved. It has been found that the presence of mitochondrial preparation increases time-dependent fluorescent response considerably and this effect depends on Ca2+ concentration in the medium. Thus, in these conditions, deesterification active probe is formed which is sensitive to Ca2+. It is shown that the accumulation of calcium ions in mitochondria in the presence of Mg-ATP and succinate depends on exogenous Ca2+ concentration and is characterized by substrate saturating. The apparent activation constant of Ca2+ accumulation is 53.9 +/- 6.9 mM, which corresponds to the physiological concentration of the cation in the cell next to mitochondria. Transit addition of Ca2+-ionophore A23187 to the incubation me- dium caused a rapid release of ionized cation from mitochondria. When proton gradient on the inner mitochondrial membrane is dissipated by protonophore CCCP, in the case of suppressing the generation of the gradient by oligomycin and in the presence of ruthenium red that inhibits Ca2+ mitochondrial accumulation systems, Ca2+ entry is significantly reduced. The results indicate the prospects of using Fluo-4 AM to study the properties of the Ca2+ accumulation system in isolated mitochondria of the myometrium.
已证实利用Ca2+敏感荧光染料Fluo-4 AM和荧光光谱法研究子宫平滑肌线粒体中能量依赖性Ca2+积累的可行性。研究发现，线粒体制剂的存在显著增加了时间依赖性荧光反应，且这种效应取决于培养基中的Ca2+浓度。因此，在这些条件下，形成了对Ca2+敏感的去酯化活性探针。结果表明，在Mg-ATP和琥珀酸存在的情况下，线粒体中钙离子的积累取决于外源Ca2+浓度，并具有底物饱和的特征。Ca2+积累的表观活化常数为53.9±6.9 mM，这与线粒体附近细胞中该阳离子的生理浓度相对应。向孵育培养基中瞬时添加Ca2+离子载体A23187会导致线粒体中离子化阳离子的快速释放。当质子载体CCCP使线粒体内膜上的质子梯度消散时，在寡霉素抑制梯度产生的情况下以及在存在抑制Ca2+线粒体积累系统的钌红的情况下，Ca2+的进入会显著减少。这些结果表明了使用Fluo-4 AM研究子宫肌层分离线粒体中Ca2+积累系统特性的前景。
