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Inhibition of the reconstituted mitochondrial oxoglutarate carrier by arginine-specific reagents.

Archives of biochemistry and biophysics (1996-07-01)
I Stipani, G Mangiullo, V Stipani, L Daddabbo, D Natuzzi, F Palmieri
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 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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2,3-Pentanedione, 97%
Sigma-Aldrich
2,3-Pentanedione, ≥96%, FCC, FG
Sigma-Aldrich
1-Phenyl-1,2-propanedione, 99%
Sigma-Aldrich
2,3-Hexanedione, ≥95%, FG
Sigma-Aldrich
2,3-Pentanedione, natural, ≥96%, FG
Sigma-Aldrich
3,4-Hexanedione, 95%
Sigma-Aldrich
2,3-Hexanedione, technical grade, 90%
Sigma-Aldrich
1-Phenyl-1,2-propanedione, 98%, FG
Sigma-Aldrich
2,3-Hexanedione, natural, 96%, FG
Supelco
2,3-Hexanedione, analytical standard