Purification of reconstitutively active alpha-oxoglutarate carrier from pig heart mitochondria.
The alpha-oxoglutarate carrier from pig heart mitochondria has been solubilized with Triton X-114 and purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. When applied to SDS gel electrophoresis, the purified protein consists of only a single protein band with an apparent Mr of 31.5 kDa. It corresponds to band 4 of the five protein bands previously identified in the hydroxyapatite pass-through of Triton X-114 solubilized heart mitochondria (Bisaccia, F. and Palmieri, F. (1984) Biochim. Biophys. Acta 766, 386-394). When reconstituted into liposomes the alpha-oxoglutarate transport protein catalyzes a phthalonate-sensitive alpha-oxoglutarate/alpha-oxoglutarate exchange. It is purified 250-fold with a recovery of 62% and a protein yield of 0.1% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., the requirements for a counteranion, the substrate specificity and the inhibitor sensitivity, are similar to those described for alpha-oxoglutarate transport in mitochondria.
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D007656:Ketoglutaric Acids / Q000378:metabolism*; D008081:Liposomes; D008293:Malates / Q000378:metabolism; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000032:analysis*; D008970:Molecular Weight; D012995:Solubility; D013552:Swine
D002352:Carrier Proteins; D003902:Detergents; D007656:Ketoglutaric Acids; D008081:Liposomes; D008293:Malates; D026901:Membrane Transport Proteins; C021723:oxoglutarate translocator