Fiermonte G, Walker J E, Palmieri F
Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
Biochem J. 1993 Aug 15;294 ( Pt 1)(Pt 1):293-9. doi: 10.1042/bj2940293.
The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been expressed at an abundant level in Escherichia coli. It accumulates in the bacterium as inclusion bodies, and none of the protein was detected in the bacterial inner membrane. The mitochondrial ADP/ATP carrier, a member of the same super-family of transport proteins as the oxoglutarate carrier, has also been expressed in E. coli. However, the expression of the ADP/ATP carrier in bacteria retards their growth, and so the levels of expression that were attained were lower than those of the oxoglutarate carrier. The oxoglutarate carrier inclusion bodies have been disaggregated with the detergent N-dodecanoyl-sarcosine, and the protein has been incorporated into liposomes. In its ability to transport oxoglutarate and malate and other known substrates of the carrier in mitochondria, and in its inhibition characteristics by a wide range of non-competitive and competitive inhibitors, this reconstituted oxoglutarate carrier is similar to the natural protein in the inner membranes of mitochondria, and to the carrier that has been purified from mitochondria and reconstituted in liposomes. These experiments remove significant obstacles to crystallization trials and to site-directed mutagenesis of the oxoglutarate carrier.
氧代戊二酸载体是牛心线粒体内膜的一种内在膜转运蛋白，已在大肠杆菌中大量表达。它以包涵体的形式在细菌中积累，在细菌内膜中未检测到该蛋白。线粒体ADP/ATP载体与氧代戊二酸载体属于同一转运蛋白超家族，也已在大肠杆菌中表达。然而，ADP/ATP载体在细菌中的表达会阻碍其生长，因此所达到的表达水平低于氧代戊二酸载体。氧代戊二酸载体包涵体已用去污剂N-十二烷基肌氨酸进行了解离，并且该蛋白已被整合到脂质体中。这种重构的氧代戊二酸载体在转运氧代戊二酸和苹果酸以及线粒体中该载体的其他已知底物的能力方面，以及在其被多种非竞争性和竞争性抑制剂抑制的特性方面，类似于线粒体内膜中的天然蛋白，也类似于从线粒体中纯化并重构到脂质体中的载体。这些实验消除了对氧代戊二酸载体进行结晶试验和定点诱变的重大障碍。
