Zernig G
Department of Pharmacology, University of Michigan, Ann Arbor 48109.
Mol Pharmacol. 1992 Dec;42(6):1010-3.
Mitochondria contain specific Ca2+ antagonist binding sites that are associated with an inner mitochondrial membrane anion channel. These mitochondrial Ca2+ antagonist receptors can be solubilized with digitonin and partially purified [as assessed by postreversible (+/-)-[3H]nitrendipine binding] using ion exchange chromatography and sucrose density gradient centrifugation. In the present study, reversible binding of the phenylakylamine Ca2+ antagonist [3H]ludopamil, an optically pure photoaffinity analog of verapamil, to the partially purified mitochondrial Ca2+ antagonist receptor complex (Kd, 9 +/- 4 microM; Bmax, 1.2 +/- 0.5 nmol/mg of protein) depended on NaNO3 and was inhibited by the 1,4-dihydropyridine niludipine and by ATP. Accordingly, the unlabeled racemic analog of [3H]ludopamil, (+/-)-LU 47781, dose-dependently inhibited the binding of the 1,4-dihydropyridine (+/-)-[3H]nitrendipine to the purified mitochondrial receptors (IC50, 2.1 +/- 0.1 microM). After UV irradiation, [3H]ludopamil specifically incorporated into two polypeptides of 12.7 +/- 0.1 kDa and 11.7 +/- 0.1 kDa, with the pharmacological profile of [3H]ludopamil photoincorporation stimulation and protection being identical to that of reversible binding.
线粒体含有与线粒体内膜阴离子通道相关的特定钙离子拮抗剂结合位点。这些线粒体钙离子拮抗剂受体可用洋地黄皂苷溶解，并通过离子交换色谱法和蔗糖密度梯度离心法进行部分纯化（通过[3H]尼群地平结合后可逆性评估）。在本研究中，苯烷基胺类钙离子拮抗剂[3H]卢帕米（维拉帕米的光学纯光亲和类似物）与部分纯化的线粒体钙离子拮抗剂受体复合物的可逆结合（解离常数Kd为9±4微摩尔；最大结合量Bmax为1.2±0.5纳摩尔/毫克蛋白质）依赖于硝酸钠，并受到1,4 - 二氢吡啶类药物尼鲁地平以及三磷酸腺苷的抑制。因此，[3H]卢帕米的外消旋未标记类似物（±）-LU 47781剂量依赖性地抑制1,4 - 二氢吡啶类药物（±）-[3H]尼群地平与纯化的线粒体受体的结合（半数抑制浓度IC50为2.1±0.1微摩尔）。紫外线照射后，[3H]卢帕米特异性地掺入两条分子量分别为12.7±0.1千道尔顿和11.7±0.1千道尔顿的多肽中，[3H]卢帕米光掺入的刺激和保护的药理学特征与可逆结合的特征相同。
