Inhibition of the reconstituted mitochondrial oxoglutarate carrier by arginine-specific reagents.
The effect of arginine-specific reagents on the function of the purified and reconstituted oxoglutarate carrier protein of the inner mitochondrial membrane has been investigated. The alpha-dicarbonyl reagents 2,3-butanedione, 2,3-pentanedione, 2,3- and 3,4-hexanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, and phenylglyoxal derivatives caused a concentration-dependent inhibition of oxoglutarate transport with an IC50 of 0.05 mM for 2,3-hexanedione, 0.08 mM for 4-hydroxy-3-nitrophenylglyoxal, and 0.17 mM for 2,3-pentanedione. The inhibition increased with pH from 6.0 to 8.0, indicating that the pK of the reacting group(s) is rather high. Mersalyl and pyridoxal 5'-phosphate (or 4,4'-dinitrostilbene-2,2'-disulfonate), which are known to react specifically and reversibly with cysteine residues and lysine residues, respectively, were unable to protect the oxoglutarate carrier against inhibition by alpha-dicarbonyl reagents. Other diketone compounds, which do not react with arginine residues, had no significant effect on the oxoglutarate transport activity. Oxoglutarate and L-malate effectively protected the oxoglutarate carrier against inactivation caused by arginine-specific reagents; other dicarboxylates, which are not substrates of the carrier, had no protective effect. A 50% substrate protection was observed at half-saturation of the external binding site. These results indicate that the arginine-specific reagents used in this investigation interact with the oxoglutarate carrier at the level of an arginine residue(s), which is essential for binding and/or translocation of substrates and which may be localized in, or near, the substrate-binding site.
Stipani
I
I
Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Italy.
Mangiullo
G
G
Stipani
V
V
Daddabbo
L
L
Natuzzi
D
D
Palmieri
F
F
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001565:Benzoates / Q000494:pharmacology; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D002417:Cattle; D047188:Chalcones; D004852:Epoxy Compounds / Q000494:pharmacology; D006588:Hexanones / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D026901:Membrane Transport Proteins; D008929:Mitochondria, Heart / Q000737:chemistry*; D010422:Pentanones / Q000494:pharmacology; D010658:Phenylglyoxal / Q000031:analogs & derivatives / Q000494:pharmacology
D001565:Benzoates; D002352:Carrier Proteins; D047188:Chalcones; D004852:Epoxy Compounds; D006588:Hexanones; D007202:Indicators and Reagents; D026901:Membrane Transport Proteins; D010422:Pentanones; C021723:oxoglutarate translocator; C043231:2,3-hexanedione; C007366:diepoxybutane; D001120:Arginine; C013186:2,3-pentanedione; D010658:Phenylglyoxal; C100349:1-phenyl-1,2-propanedione