The mitochondrial oxoglutarate carrier: sulfhydryl reagents bind to cysteine-184, and this interaction is enhanced by substrate binding.
The interaction of sulfhydryl reagents with the oxoglutarate carrier (OGC) of bovine heart mitochondria was investigated in proteoliposomes reconstituted from purified carrier and lipids. Incubation of the proteoliposomes with maleimides or mercurials led to inhibition of the oxoglutarate carrier protein. The inhibition of oxoglutarate transport by mercurials was removed by dithioerythritol (DTE), whereas inhibition by maleimides was not. Preincubation of the proteoliposomes with mercurials protected the carrier protein against inactivation by the fluorescent sulfhydryl reagent N-(1-pyrenyl)maleimide (PM) and decreased the fluorescence associated with the carrier, indicating that mercurials bind to the same cysteine which is modified by PM. The presence of the substrates oxoglutarate and malate increased the binding of PM to the reconstituted carrier as well as the degree of inhibition of the reconstituted transport activity caused by PM, other maleimides, and mercurials. This result is consistent with the assumption that substrate binding causes a change in the tertiary structure of the carrier protein. The primary sequence of the oxoglutarate carrier contains three cysteines (Cys-184, Cys-221, and Cys-224). We provide evidence that PM labels only Cys-184, whereas Cys-221 and Cys-224 are linked by a disulfide bridge.
Capobianco
L
L
Department of Pharmaco-Biology, University of Bari, Italy.
Bisaccia
F
F
Mazzeo
M
M
Palmieri
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine; D004305:Dose-Response Relationship, Drug; D007656:Ketoglutaric Acids / Q000494:pharmacology; D008293:Malates / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D026901:Membrane Transport Proteins; D017669:Mercury Compounds / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000378:metabolism*; D008969:Molecular Sequence Data; D011510:Proteolipids / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002352:Carrier Proteins; D007656:Ketoglutaric Acids; D008293:Malates; D008301:Maleimides; D026901:Membrane Transport Proteins; D017669:Mercury Compounds; D011510:Proteolipids; D013439:Sulfhydryl Reagents; C021723:oxoglutarate translocator; C020485:proteoliposomes; C030298:malic acid; C003944:N-(3-pyrene)maleimide; D003545:Cysteine