Dawson A P, Rich G T, Loomis-Husselbee J W
School of Biological Sciences, University of East Anglia, Norwich, U.K.
Biochem J. 1995 Sep 1;310 ( Pt 2)(Pt 2):371-4. doi: 10.1042/bj3100371.
The rate of unidirectional efflux of 45Ca from rat liver microsomal vesicles loaded with 45Ca and then treated with thapsigargin is not inhibited by increased [Ca2+] in the external medium, although the net efflux rate is substantially inhibited. We have used this property to measure the electrochemical gradient of Ca2+ from the inside to the outside of the vesicles at a series of Ca2+ loadings, by measuring the external [Ca2+]free at which there is zero net efflux. At a loading of 7.9 +/- 0.6 nmol/mg of microsomal protein, the apparent internal [Ca2+]free is 21 +/- 1.6 microM. As the loading is increased, the internal [Ca2+]free increases linearly up to a value of 47 +/- 3.6 microM at a loading of 21.6 +/- 1.6 nmol/mg. Using a similar technique, the value for [Ca2+]free in the endoplasmic reticulum of permeabilized L1210 cells was found to be 12.5 microM.
用毒胡萝卜素处理预先装载了⁴⁵Ca的大鼠肝脏微粒体囊泡后，尽管外部介质中[Ca²⁺]升高会显著抑制⁴⁵Ca的净流出速率，但从这些囊泡中⁴⁵Ca的单向流出速率却不受影响。我们利用这一特性，通过测量净流出为零时的外部游离[Ca²⁺]，在一系列Ca²⁺装载量下测定了从囊泡内部到外部的Ca²⁺电化学梯度。在微粒体蛋白装载量为7.9±0.6 nmol/mg时，表观内部游离[Ca²⁺]为21±1.6 μM。随着装载量增加，内部游离[Ca²⁺]线性增加，在装载量为21.6±1.6 nmol/mg时达到47±3.6 μM。使用类似技术，发现通透的L1210细胞内质网中的游离[Ca²⁺]值为12.5 μM。
