Indiveri C, Palmieri F, Bisaccia F, Krämer R
Biochim Biophys Acta. 1987 Mar 4;890(3):310-8. doi: 10.1016/0005-2728(87)90158-7.
The 2-oxoglutarate carrier from the inner membrane of bovine heart mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted with egg yolk phospholipid vesicles by the freeze-thaw-sonication technique. In the reconstituted system the incorporated 2-oxoglutarate carrier catalyzed a first-order reaction of 2-oxoglutarate/2-oxoglutarate exchange. The substrate affinity for 2-oxoglutarate was determined to be 65 +/- 18 microM (15 determinations) and the maximum exchange rate at 25 degrees C reaches 4000-22,000 mumol/min per g protein, in dependence of the particular reconstitution conditions. The activation energy of the exchange reaction is 54.3 kJ/mol. The transport is independent of pH in the range between 6 and 8. When the first fraction of the hydroxyapatite/celite column eluate was used for reconstitution, besides the 2-oxoglutarate/2-oxoglutarate exchange, a significant activity of unidirectional uptake was observed. This activity may be due to a population of the carrier protein which is in a different state.
通过羟基磷灰石/硅藻土柱色谱法对牛心线粒体内膜中的2-氧代戊二酸载体进行了纯化，并采用冻融-超声处理技术将其与蛋黄磷脂囊泡进行了重组。在重组系统中，掺入的2-氧代戊二酸载体催化了2-氧代戊二酸/2-氧代戊二酸的一级交换反应。测定2-氧代戊二酸的底物亲和力为65±18μM（15次测定），在25℃下，最大交换速率根据具体的重组条件可达4000 - 22000μmol/（min·g蛋白）。交换反应的活化能为54.3 kJ/mol。在pH值6至8的范围内，转运不受pH影响。当使用羟基磷灰石/硅藻土柱洗脱液的第一部分进行重组时，除了2-氧代戊二酸/2-氧代戊二酸交换外，还观察到明显的单向摄取活性。这种活性可能归因于处于不同状态的载体蛋白群体。
