Bisaccia F, De Palma A, Prezioso G, Palmieri F
Department of Pharmaco-Biology, University of Bari, Italy.
Biochim Biophys Acta. 1990 Sep 19;1019(3):250-6. doi: 10.1016/0005-2728(90)90201-e.
The tricarboxylate carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatography on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-saturation constant was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the maximum exchange rate at 25 degrees C reached 2000 mumols/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).
通过在羟基磷灰石/硅藻土上进行色谱法，从大鼠肝脏线粒体中纯化了三羧酸载体，并通过在Amberlite上进行疏水色谱法去除去污剂，将其重构成磷脂囊泡。通过使用0.8的Triton X-114/磷脂比例、6%的心磷脂以及通过单个Amberlite柱进行24次传代，可获得最佳转运活性。在重构系统中，掺入的三羧酸载体催化了柠檬酸/柠檬酸或柠檬酸/苹果酸交换的一级反应。交换反应的活化能为70.1 kJ/mol。交换速率的最适pH在7至8之间。柠檬酸的半饱和常数为0.13 mM，苹果酸的半饱和常数为0.76 mM。所有这些性质都与完整线粒体中三羧酸转运系统所描述的性质相似。在蛋白脂质体中，25℃时的最大交换速率达到每克蛋白质2000 μmol/min。该值与存在于脂质体外部或内部空间的底物类型（柠檬酸或苹果酸）无关。
