Abstract
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 α-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 IC50of 0.05 mMfor 2,3-hexanedione, 0.08 mMfor 4-hydroxy-3-nitrophenylglyoxal, and 0.17 mMfor 2,3-pentanedione. The inhibition increased with pH from 6.0 to 8.0, indicating that the pKof 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 α-dicarbonyl reagents. Other diketone compounds, which do not react with arginine residues, had no significant effect on the oxoglutarate transport activity. Oxoglutarate andL-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.
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