Identification and purification of the reconstitutively active glutamine carrier from rat kidney mitochondria.
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.
Indiveri
C
C
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, 70125 Bari, Italy.
Abruzzo
G
G
Stipani
I
I
Palmieri
F
F
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
D002352:Carrier Proteins; D003902:Detergents; D008081:Liposomes; C037853:glutamine transport proteins; D005973:Glutamine; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D011732:Pyridoxal Phosphate; D005033:Ethylmaleimide