Yoon P S, Sharp R R
Biochemistry. 1985 Dec 3;24(25):7269-73. doi: 10.1021/bi00346a037.
High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.
高分辨率质子核磁共振光谱已被用于监测在钙离子通过膜流入过程中嗜铬粒蛋白颗粒空泡的内部pH值。为此，通过在含有乙二胺四乙酸二钠络合物（Na2.EDTA）的介质中裂解并重新封闭嗜铬粒蛋白颗粒来制备空泡。未络合的EDTA和Ca.EDTA在质子核磁共振光谱中产生不同的亚甲基峰组。在氘代介质中，游离EDTA的滴定pK接近6.6；伴随滴定的化学位移已被用于监测嗜铬粒蛋白颗粒空泡内部由于ATP酶活性以及在钙离子流入和络合物形成过程中EDTA的去质子化而发生的囊泡内pH值变化。ATP酶活性导致一个可通过核磁共振检测到的质子梯度，该梯度可被尼日利亚菌素消散。监测钙离子摄取的实验表明，由于Ca.EDTA络合物形成而在空泡内部释放的质子不会通过与钙离子进入相关的过程从空泡中排出。这表明嗜铬粒蛋白颗粒膜的钙离子转运系统在没有同时进行质子反向转运的情况下发生，并且在能量上不直接与跨膜pH梯度耦合。
