Indiveri C, Dierks T, Krämer R, Palmieri F
Department of Pharmaco-Biology, University of Bari, Italy.
Eur J Biochem. 1991 Jun 1;198(2):339-47. doi: 10.1111/j.1432-1033.1991.tb16021.x.
The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically. A complete set of half-saturation constants (Km) was established for the two different substrates oxoglutarate and malate on both the external and the internal sides of the membrane. The internal affinities for oxoglutarate (Km 0.17 mM) and malate (Km 0.7 mM) were higher than the corresponding external affinities (Km 0.3 mM and 1.4 mM, respectively). The exclusive presence of a single transport affinity for each substrate on one side of the membrane indicated a unidirectional insertion of the oxoglutarate carrier into the liposomal membrane. The Km values and also the maximum exchange rates (8-11 mumol.min-1.mg protein-1) for oxoglutarate and malate were independent of the nature of the counter substrate on the other side of the membrane. Under these defined conditions we analyzed the antiport mechanism in two-reactant initial velocity studies varying both the internal and external substrate concentrations. From the kinetic patterns obtained, a sequential type of mechanism became evident, implying that one internal and one external substrate molecule form a ternary complex with the carrier before transport occurs. A quantitative analysis of substrate interaction with the unloaded or single-substrate-occupied carrier revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrate. This kinetic mechanism agrees with previous results obtained in intact mitochondria. Considering also the data available for other mitochondrial carriers, a common kinetic mechanism (sequential type) for this carrier family is suggested.
对从牛心线粒体中纯化得到的重组草酰戊二酸载体的转运机制进行了动力学研究。针对膜外侧和内侧的两种不同底物草酰戊二酸和苹果酸，确定了一套完整的半饱和常数（Km）。草酰戊二酸（Km 0.17 mM）和苹果酸（Km 0.7 mM）的内侧亲和力高于相应的外侧亲和力（分别为Km 0.3 mM和1.4 mM）。膜一侧每种底物仅存在单一转运亲和力，这表明草酰戊二酸载体单向插入脂质体膜中。草酰戊二酸和苹果酸的Km值以及最大交换速率（8 - 11 μmol·min⁻¹·mg蛋白⁻¹）与膜另一侧反向底物的性质无关。在这些限定条件下，我们在改变内侧和外侧底物浓度的双反应物初始速度研究中分析了反向转运机制。从获得的动力学模式来看，一种顺序型机制变得明显，这意味着在转运发生之前，一个内侧和一个外侧底物分子与载体形成三元复合物。对底物与空载或单底物占据载体相互作用的定量分析表明，满足快速平衡随机条件，其特征是内侧和外侧底物快速且独立地结合。这种动力学机制与之前在完整线粒体中获得的结果一致。考虑到其他线粒体载体的现有数据，建议该载体家族具有共同的动力学机制（顺序型）。
