ROLE OF CALCIUM IN THE REGULATION OF ENERGY METABOLISM

该项目旨在评估 钙离子在能量代谢中的调节作用。 我们有 应用荧光螯合剂方案， 开发用于测量胞质游离Ca 2+， 线粒体基质游离Ca（（Ca 2+））m的测量。 这 使我们能够直接研究（Ca 2+）m和 基质酶丙酮酸脱氢酶的激活， 首次在离体大鼠心肌线粒体负载的研究中 Indo-1 发现活化的K0.5为300 nM Ca 2： 该值不受程序的影响，例如省略Mg 2+， Na+，其改变Ca浓度梯度的大小 穿过线粒体膜。 我们还研究了 丙酮酸脱氢酶的激活率 电刺激离体豚鼠心脏： 平行研究中，线粒体迅速从刺激的 心脏，使用一个协议，防止损失或获得的 线粒体钙，使我们能够将这个数量与水平 脱氢酶的激活。 由于脱氢酶的重要性 激活在于NADH/NAD比率，其可以维持在 面对工作量的增加，我们也测量了 饱和培养大鼠心肌线粒体中的NADH/NAD 和非饱和浓度的丙酮酸或2-酮戊二酸， 呼吸底物，在存在或不存在Ca 2+的情况下，和 将其与氧化磷酸化的通量相关联。 最后，我们检查了心肌线粒体的能力， 衰老的狗氧化底物，重点是脂肪酸） 作为生活方式的函数，即久坐与锻炼。

This project is designed to assess the physiological importance of calcium ions in the regulation of energy metabolism. We have applied the fluorescent chelating-agent protocol, which was developed for the measurement cytosolic free Ca2+, to the measurement of mitochondrial matrix free Ca ((Ca2+))m. This allowed us to study directly the relationship between (Ca2+)m and the activation of the matrix enzyme pyruvate dehydrogenase for the first time in studies with isolated rat cardiac mitochondria loaded with indo-1. The KO.5 for activation was found to be 300 nM Ca2: this value was unaffected by procedures, e.g. omission of Mg2+, Na+, which change the magnitude of the Ca concentration gradient across the mitochondrial membrane. We have also studied the degree of activation of pyruvate dehydrogenase as a function of the rate of electrical stimulation of isolated guinea pig hearts: in a parallel study, mitochondria were rapidly isolated from stimulated hearts, using a protocol which prevents loss or gain of mitochondrial Ca and allows us to relate this quantity to the level of dehydrogenase activation. As the significance of dehydrogenase activation lies in the NADH/NAD ratio which can be maintained in the face of elevated work -loads, we have also measured the ratio NADH/NAD in suspensions of rat heart mitochondria with saturating and non-saturating concentrations of pyruvate or 2-oxoglutarate as respiratory substrate, in the presence or absence of Ca2+, and correlated this with the flux through oxidative phosphorylation. Finally, we have examined the ability of cardiac mitochondria from senescent dogs to oxidize substrates with emphasis on fatty acids) as a function of lifestyle, viz-sedentary versus exercised.