| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000)
|
|---|
| Research Abstract |
The mitochondrial membrane is impermeable to small molecules except for the selected metabolites such as substrates and products of oxidative phosphorylation. Inner mitochondrial membrane has special machineries for the transport of small molecules, i.e. ADP, ATP, malate, 2-oxoglutarate, aspartate and glutamate. On such machinery, malate-aspartate shuttle, consists of translocators for these dicarboxylates, in addition to cytosolic and mitochondrial isozymes of both aspartate aminotransferase and malate dehydrogenase. Here we report a method for isolation of these translocators (carrier proteins). First, we isolated mitochondrial particles from pig heart muscle as a source of these carrier proteins. The 2-oxoglutarate carrier from the membrane of pig heart mitochondria was purified by a combination of hydroxyapatite and ion-exchange chromatography. The transport activity was measured by incorporating the carrier protein samples into phospholipid vesicles by the freeze-thaw-sonication method. Antibodies to 2-oxoglutarate carrier were obtained for the purpose of screening of the expression vector for the carrier protein. However, titers of antibody were not sufficiently high for the use. On the other hand, the aspartate carrier was solubilized with detergent and partially purified from pig heart mitochondria. Extensive purification was hampered by its instability encountered during the purification procedures. An application of affinity chromatography techniques was attempted. However, we could not find effective affinity ligands for the aspartate carrier protein.
|
