Joseph S K, Coll K E, Cooper R H, Marks J S, Williamson J R
J Biol Chem. 1983 Jan 25;258(2):731-41.
The steady state relationship between intra- and extramitochondrial free Ca2+ across the inner mitochondrial membrane has been investigated in isolated liver mitochondria. The extramitochondrial free Ca2+ concentration was essentially independent of the mitochondrial calcium content above 4 nmol/mg of protein. Below this value, a decrease in the mitochondrial calcium content was accompanied by a decrease in the extramitochondrial free Ca2+ concentration. The experimental data are compatible with a model in which the steady state distribution of calcium is described in terms of the kinetic parameters of the separate carriers catalyzing Ca2+ influx and efflux across the mitochondrial inner membrane. The corresponding relationship between cytosolic free Ca2+ concentration and the amounts of calcium in the mitochondria and endoplasmic reticulum was investigated in isolated river cells over a range of cellular Ca2+ contents by using a nondisruptive technique based on the selective release of calcium from mitochondrial and total cellular pools by addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and A23187, respectively. A net increase in cell calcium from 1 to 5 nmol/mg dry weight, increased the cytosolic free Ca2+ concentration from 0.1 to about 0.3 microM and increased the calcium contents of both mitochondria and endoplasmic reticulum. Above 5 nmol of calcium/mg cell dry weight, the endoplasmic reticulum calcium pool became filled, and further increases in calcium content were accounted for by increases of the mitochondrial pool but no further increase of the cytosolic free Ca2+ concentration. These studies and experiments with mixtures of isolated microsomes and mitochondria suggest that, in cells as normally isolated (containing 5 to 6 nmol of calcium/mg dry weight), the endoplasmic reticulum is saturated with calcium and is unlikely to play a major role as an intracellular calcium buffer. The in situ mitochondrial calcium content is sufficiently high (approximately 16 nmol/mg of protein) for these organelles to buffer effectively the cytosolic free Ca2+ concentration at a value of about 0.3 microM. In addition, it may be concluded that intramitochondrial Ca2+-dependent enzymes will be exposed to saturating concentrations of free Ca2+.
已在分离的肝线粒体中研究了线粒体内外游离Ca2+跨线粒体内膜的稳态关系。当线粒体钙含量高于4 nmol/mg蛋白质时，线粒体外游离Ca2+浓度基本与线粒体钙含量无关。低于该值时，线粒体钙含量的降低伴随着线粒体外游离Ca2+浓度的降低。实验数据与一个模型相符，在该模型中，钙的稳态分布是根据催化Ca2+流入和流出线粒体内膜的各个载体的动力学参数来描述的。通过一种基于分别添加羰基氰对三氟甲氧基苯腙和A23187从线粒体和总细胞池中选择性释放钙的非破坏性技术，在一系列细胞Ca2+含量范围内，研究了分离的肝细胞中胞质游离Ca2+浓度与线粒体和内质网中钙含量之间的相应关系。细胞钙含量从1 nmol/mg干重净增加到5 nmol/mg干重，使胞质游离Ca2+浓度从0.1 microM增加到约0.3 microM，并增加了线粒体和内质网中的钙含量。当钙含量高于5 nmol/mg细胞干重时，内质网钙池被填满，钙含量的进一步增加是由线粒体池的增加引起的，但胞质游离Ca2+浓度没有进一步增加。这些研究以及对分离的微粒体和线粒体混合物进行的实验表明，在正常分离的细胞（含有5至6 nmol钙/mg干重）中，内质网钙已饱和，不太可能作为细胞内钙缓冲剂发挥主要作用。原位线粒体钙含量足够高（约16 nmol/mg蛋白质），这些细胞器能够有效地将胞质游离Ca2+浓度缓冲在约0.3 microM的值。此外，可以得出结论，线粒体内依赖Ca2+的酶将暴露于游离Ca2+的饱和浓度。
