Nicholls D G, Sihra T S, Sanchez-Prieto J
J Neurochem. 1987 Jul;49(1):50-7. doi: 10.1111/j.1471-4159.1987.tb03393.x.
An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.
本文描述了一种酶联荧光测定法，用于连续监测豚鼠突触体中谷氨酸的单向外流。新鲜悬浮的极化突触体中谷氨酸的外流速率为0.35 - 0.39 nmol min⁻¹ mg蛋白质⁻¹，且不受细胞外Ca²⁺的显著影响。在无Ca²⁺的情况下，30 mM KCl去极化可使该速率翻倍，而在有Ca²⁺的情况下，该测定法的初始动力学与最初5 s内释放0.6 nmol mg蛋白质⁻¹一致。Ca²⁺依赖性释放的最终程度达到1.9 nmol mg蛋白质⁻¹，占突触体内谷氨酸总含量的8.5%。在去极化前将突触体于30℃预孵育2 h会导致非Ca²⁺依赖性释放减少，Ca²⁺依赖性释放增加，这与氨基酸在突触体内的重新定位一致。
