title: Identification and purification of the reconstitutively active glutamine carrier from rat kidney mitochondria.
abstract: The glutamine carrier from rat kidney mitochondria, solubilized in dodecyl octaoxyethylene ether (C12E8) and partly purified on hydroxyapatite, was identified and completely purified by Celite chromatography. On SDS/PAGE, the purified glutamine carrier consisted of a single protein band with an apparent molecular mass of 41.5 kDa. When reconstituted into liposomes, the glutamine carrier catalysed both the unidirectional flux of glutamine and the glutamine/glutamine countertransport, which were completely inhibitable by a mixture of pyridoxal 5'-phosphate and N-ethylmaleimide. The carrier protein was purified 474-fold with a recovery of 58% and a protein yield of 0.12% with respect to the mitochondrial extract. The glutamine carrier-mediated transport is quite specific for l-glutamine. l-Asparagine is the only other amino acid that is efficiently transported by the reconstituted carrier protein. d-Glutamine, l-glutamate and l-aspartate are very poor substrates. The transport activity was inhibited by several thiol-group and amino-group reagents.
authors.affiliation: Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, 70125 Bari, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Indiveri
authors.forename: G
authors.initials: G
authors.lastname: Abruzzo
authors.forename: I
authors.initials: I
authors.lastname: Stipani
authors.forename: F
authors.initials: F
authors.lastname: Palmieri
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005033:Ethylmaleimide / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000378:metabolism; D051381:Rats; D012995:Solubility
chemical_list: D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; C037853:glutamine transport proteins; D005973:Glutamine; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D011732:Pyridoxal Phosphate; D005033:Ethylmaleimide