Jacobus W E, Tiozzo R, Lugli G, Lehninger A L, Carafoli E
J Biol Chem. 1975 Oct 10;250(19):7863-70.
When intact rat heart mitochondria were pulsed with 150 nmol of CaCl2/mg of mitochondrial protein, only a marginal stimulation of the rate of oxygen consumption was observed. This result was obtained with mitochondria isolated in either the presence or absence of nagarse. In contrast, rat liver mitochondria under similar conditions demonstrated a rapid, reversible burst of respiration associated with energy-linked calcium accumulation. Direct analysis of calcium retention using 45Ca and Millipore filtration indicated that calcium was accumulated by heart mitochondria under the above conditions via a unique energy-dependent process. The rate of translocation by heart mitochondria was less than that of liver mitochondria; likewise the release of bound calcium back into the medium was also retarded. These results suggest that the slower accumulation and release of calcium is characteristic of heart mitochondria. The amound of calcium bound was independent of penetrant anions at low calcium concentrations. Above 100 nmol/mg of mitochondrial protein, the total calcium bound was increased by the presence of inorganic phosphate. Under nonrespiring conditions, a biphasic Scatchard plot indicative of binding sites with different affinities for Ca2+ was observed. The extrapolated constants are 7.5 nmol/mg bound with an apparent half-saturation value of 75 muM and 42.5 nmol/mg bound with half-saturation at 1.15 mM. The response of the reduced State 4 cytochrome b to pulsed additions of Ca2+ was used to calculate an energy-dependent half-saturation constant of 40 muM. When the concentration of free calcium was stabilized at low levels with Ca2+-EGTA buffers, the spectrophotometrically determined binding constant decreased two orders of magnitude to an apparent affinity of 4.16 X 10(-7) M. Primary of calcium transport over oxidative phosphorylation was not observed with heart mitochondria. The phosphorylation of ADP competed with Ca2+ accumulation, depressed the rates of cation transport, and altered the profile of respiration-linked H+ movements. Consistent with these result was the observation that with liver mitochondrial the magnitude of the cytochrome b oxidation-reduction shift was greater for Ca2+ than for ADP, whereas calcium responses never surpassed the ADP response in heart mitochondria. Furthermore, Mg2+ ingibited calcium accumulation by heart mitochondria while having only a slight effect upon calcium transport in liver mitochondria. The unique energetics of heart mitochondrial calcium transport are discussed relative to the regulated flux of cations during the cardiac excitation-relaxation cycle.
当向完整的大鼠心脏线粒体中加入150 nmol氯化钙/毫克线粒体蛋白进行脉冲处理时，仅观察到氧消耗速率有轻微刺激。无论在有或没有纳加酶的情况下分离线粒体，均得到此结果。相比之下，在类似条件下的大鼠肝脏线粒体表现出与能量相关的钙积累相关的快速、可逆的呼吸爆发。使用45Ca和密理博过滤对钙保留进行直接分析表明，在上述条件下，心脏线粒体通过独特的能量依赖过程积累钙。心脏线粒体的转运速率低于肝脏线粒体；同样，结合钙释放回培养基中的过程也受到阻碍。这些结果表明，钙积累和释放较慢是心脏线粒体的特征。在低钙浓度下，结合的钙量与渗透阴离子无关。线粒体蛋白含量超过100 nmol/毫克时，无机磷酸盐的存在会增加总钙结合量。在非呼吸条件下，观察到双相Scatchard图，表明对Ca2+具有不同亲和力的结合位点。外推常数为7.5 nmol/毫克，表观半饱和值为75 μM，以及42.5 nmol/毫克，半饱和值为1.15 mM。利用还原态细胞色素b对Ca2+脉冲添加的响应来计算能量依赖的半饱和常数为40 μM。当用Ca2+-EGTA缓冲液将游离钙浓度稳定在低水平时，分光光度法测定的结合常数下降两个数量级，表观亲和力为4.16×10(-7) M。未观察到心脏线粒体钙转运优先于氧化磷酸化。ADP的磷酸化与Ca2+积累竞争，降低阳离子转运速率，并改变呼吸相关H+运动的模式。与这些结果一致的是，观察到对于肝脏线粒体，Ca2+引起的细胞色素b氧化还原变化幅度大于ADP，而在心脏线粒体中钙反应从未超过ADP反应。此外，Mg2+抑制心脏线粒体的钙积累，而对肝脏线粒体的钙转运只有轻微影响。相对于心脏兴奋-松弛周期中阳离子的调节通量，讨论了心脏线粒体钙转运的独特能量学。
