Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria.
The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D005971:Glutamates / Q000378:metabolism*; D007425:Intracellular Membranes / Q000032:analysis; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification*; D008929:Mitochondria, Heart / Q000032:analysis*; D008970:Molecular Weight; D012995:Solubility
D002352:Carrier Proteins; D005971:Glutamates; D008081:Liposomes; D008565:Membrane Proteins; D001224:Aspartic Acid