Hansford R G, Castro F
Biochem J. 1985 Apr 1;227(1):129-36. doi: 10.1042/bj2270129.
The steady-state content of active (dephospho) pyruvate dehydrogenase (PDHA) of suspensions of coupled rat brain mitochondria oxidizing succinate was found to be markedly increased with increasing free Ca2+ ion concentration of the medium, with a half-maximal effect at 10(-6.43) M Ca2+. Other ions were present in these studies at concentrations appropriate for the cytosol. Depolarization of the plasma membrane of synaptosomes caused an increase in the steady-state content of PDHA, with veratridine giving a larger increase than depolarization by 33 mM-KCl. Values were 68 +/- 1% (n = 13) and 81 +/- 1% (n = 19) of maximal activity, for control incubations and incubations in the presence of 30 microM-veratridine, respectively. Measurements of cytosolic free Ca2+ concentrations ([Ca2+]cyt.) in these suspensions of synaptosomes, with the use of the fluorescent Ca2+-indicator Quin-2, indicated an increase on depolarization, with the change due to 30 microM-veratridine being larger in extent than that due to 33 mM-KCl. Values were 217 +/- 21 nM (n = 15), 544 +/- 48 nM (n = 15) and 783 +/- 75 nM (n = 14) for control, KCl-depolarized and veratridine-depolarized synaptosomes respectively. Experiments in which synaptosomes were treated with Ruthenium Red, an inhibitor of mitochondrial Ca2+ uptake, gave much lower resting contents of PDHA (42 +/- 2% of maximal), but failed to prevent totally an increase on depolarization. Addition of an excess of EGTA to the synaptosomal suspension just before the addition of veratridine resulted in a partial diminution in the response of PDHA content. Parallel studies with Quin-2 indicated no increase in [Ca2+]cyt. on addition of veratridine, under these conditions. Thus an increase in [Ca2+]cyt. forms only a part of the mechanism whereby pyruvate dehydrogenase interconversion responds to depolarization. A decrease in the ATP/ADP ratio may also be important, as inferred from the results of experiments with ouabain, which inhibits the Na+ + K+-dependent ATPase.
在氧化琥珀酸的大鼠脑线粒体偶联悬浮液中，活性（去磷酸化）丙酮酸脱氢酶（PDHA）的稳态含量随着培养基中游离Ca2+离子浓度的增加而显著增加，在Ca2+浓度为10(-6.43)M时达到最大效应的一半。在这些研究中，其他离子的浓度与细胞质中的浓度相当。突触体细胞膜的去极化导致PDHA的稳态含量增加，藜芦碱引起的增加比33 mM - KCl引起的去极化更大。在对照孵育和存在30 microM - 藜芦碱的孵育中，活性值分别为最大活性的68 +/- 1%（n = 13）和81 +/- 1%（n = 19）。使用荧光Ca2+指示剂Quin - 2测量这些突触体悬浮液中的细胞质游离Ca2+浓度（[Ca2+]cyt.），结果表明去极化时浓度增加，30 microM - 藜芦碱引起的变化程度大于33 mM - KCl引起的变化。对照、KCl去极化和藜芦碱去极化的突触体的[Ca2+]cyt.值分别为217 +/- 21 nM（n = 15）、544 +/- 48 nM（n = 15）和783 +/- 75 nM（n = 14）。用线粒体Ca2+摄取抑制剂钌红处理突触体的实验中，PDHA的静息含量低得多（为最大活性的42 +/- 2%），但未能完全阻止去极化时的增加。在加入藜芦碱之前，向突触体悬浮液中加入过量的EGTA会导致PDHA含量的反应部分减弱。在这些条件下，用Quin - 2进行的平行研究表明，加入藜芦碱后[Ca2+]cyt.没有增加。因此，[Ca2+]cyt.的增加只是丙酮酸脱氢酶相互转化对去极化反应机制的一部分。从哇巴因实验结果推断，ATP/ADP比值的降低也可能很重要，哇巴因可抑制Na+ + K+依赖性ATP酶。
