RESUMO
Using a functional mitochondrial oxoglutarate carrier mutant devoid of Cys residues (C-less carrier), each amino acid residue in transmembrane domain IV and flanking hydrophilic loops (from T179 to S205) was replaced individually with Cys. The great majority of the 27 mutants exhibited significant oxoglutarate transport in reconstituted liposomes as compared to the activity of the C-less carrier. In contrast, Cys substitution for G183, R190, Q198, and Y202, in either C-less or wild-type carriers, yielded molecules with complete loss of oxoglutarate transport activity. G183 and R190 could be partially replaced only by Ala and Lys, respectively, whereas Q198 and Y202 were irreplaceable with respect to oxoglutarate transport. Of the single-Cys mutants tested, only T187C, A191C, V194C, and N195C were strongly inactivated by N-ethylmaleimide and by low concentrations of methanethiosulfonate derivatives. Oxoglutarate protects Cys residues at positions 187, 191, and 194 against reaction with N-ethylmaleimide. These positions as well as the residues found to be essential for the carrier activity, except Y202 which is located in the extramembrane loop IV-V, reside on the same face of transmembrane helix IV, probably lining part of a water-accessible crevice or channel between helices of the oxoglutarate carrier.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cisteína/genética , Metanossulfonato de Etila/análogos & derivados , Ácidos Cetoglutáricos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Mutagênese Sítio-Dirigida , Reagentes de Sulfidrila/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Proteínas de Transporte/antagonistas & inibidores , Bovinos , Metanossulfonato de Etila/farmacologia , Etilmaleimida/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Mesilatos/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Proteolipídeos/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismoRESUMO
The effect of pyridoxal 5'-phosphate and some other lysine reagents on the purified, reconstituted mitochondrial oxoglutarate transport protein has been investigated. The inhibition of oxoglutarate/oxoglutarate exchange by pyridoxal 5'-phosphate can be reversed by passing the proteoliposomes through a Sephadex column but the reduction of the Schiff's base by sodium borohydride yielded an irreversible inactivation of the oxoglutarate carrier protein. Pyridoxal 5'-phosphate, which caused a time- and concentration-dependent inactivation of oxoglutarate transport with an IC50 of 0.5 mM, competed with the substrate for binding to the oxoglutarate carrier (Ki = 0.4 mM). Kinetic analysis of oxoglutarate transport inhibition by pyridoxal 5'-phosphate indicated that modification of a single amino acid residue/carrier molecule was sufficient for complete inhibition of oxoglutarate transport. After reduction with sodium borohydride [3H]pyridoxal 5'-phosphate bound covalently to the oxoglutarate carrier. Incubation of the proteoliposomes with oxoglutarate or L-malate protected the carrier against inactivation and no radioactivity was found associated with the carrier protein. In contrast, glutarate and substrates of other mitochondrial carrier proteins were unable to protect the carrier. Mersalyl, which is a known sulfhydryl reagent, also failed to protect the oxoglutarate carrier against inhibition by pyridoxal 5'-phosphate. These results indicate that pyridoxal 5'-phosphate interacts with the oxoglutarate carrier at a site(s) (i.e., a lysine residue(s) and/or the amino-terminal glycine residue) which is essential for substrate translocation and may be localized at or near the substrate-binding site.
