Assimacopoulos-Jeannet F, McCormack J G, Jeanrenaud B
J Biol Chem. 1986 Jul 5;261(19):8799-804.
Mitochondria were prepared by a method including a Percoll purification step after the rapid homogenization of livers of fed rats which had been perfused either under unstimulated conditions or in the presence of vasopressin and/or glucagon. The two hormones separately or together increased the total calcium content of the mitochondria. This enhancement was accompanied by parallel increases in activities of the Ca2+-sensitive intramitochondrial enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The effects of the two hormones on total mitochondrial calcium and on the activities of the oxidative enzymes were additive. The persistent enhancements of mitochondrial calcium content and enzyme activities were partially reversed by the addition of Na+ ions to the mitochondrial incubations; these effects of Na+ were blocked by diltiazem, a selective inhibitor of Na+-induced Ca2+ release. Mitochondria from control livers were incubated in vitro with CaCl2 to achieve various calcium content, and mitochondrial enzyme activities and calcium content were measured. A good correlation was obtained between the total calcium content and the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase. The results obtained are consistent with the hypothesis that vasopressin and glucagon additively cause increases in intramitochondrial [Ca2+] and so bring about the activations of these key enzymes of mitochondrial oxidative metabolism.
线粒体的制备方法是,先将喂食后的大鼠肝脏在未受刺激的条件下,或在存在血管加压素和/或胰高血糖素的情况下进行灌注,然后迅速匀浆,再经过一个Percoll纯化步骤。这两种激素单独或共同作用时,都会增加线粒体的总钙含量。这种增加伴随着线粒体中对Ca2+敏感的酶——丙酮酸脱氢酶和2-氧代戊二酸脱氢酶活性的平行增加。这两种激素对线粒体总钙含量和氧化酶活性的影响是相加的。通过向线粒体孵育液中添加Na+离子,可部分逆转线粒体钙含量和酶活性的持续增加;Na+的这些作用被地尔硫䓬阻断,地尔硫䓬是一种Na+诱导的Ca2+释放的选择性抑制剂。将对照肝脏的线粒体在体外与CaCl2一起孵育,以达到不同的钙含量,并测量线粒体酶活性和钙含量。在总钙含量与丙酮酸脱氢酶和2-氧代戊二酸脱氢酶的活性之间获得了良好的相关性。所得结果与以下假设一致:血管加压素和胰高血糖素相加会导致线粒体内[Ca2+]增加,从而引起线粒体氧化代谢的这些关键酶的激活。